Antibodies Against HPA-1a

ABSTRACT

Provided is an isolated antibody that specifically binds to HPA-1a. Also provided is a nucleic acid molecule that encodes the antibody, and compositions comprising the antibody. Also provided is a method of producing the antibody and methods and uses which employ the antibody. Also provided are therapeutic uses of the antibody, for example in the treatment or prophylaxis of fetal and neonatal alloimmune thrombocytopenia (FNAIT).

Priority is claimed under 35 U.S.C. § 119 to British Application Nos.1405775.6 filed on Mar. 31, 2014 and 1417614.3, filed on Oct. 6, 2014,respectively, and under 35 U.S.C. § 365 to PCT/EP20151057102 filed onMar. 31, 2015.

The present invention relates generally to the field of antibodies, inparticular to antibodies against HPA-1a (human platelet antigen-1a ).The invention further relates to compositions and immunoconjugatescomprising such antibodies and to methods of producing such antibodies.The invention also relates to methods of detecting for the presence orabsence of HPA-1a in a sample and methods for the treatment,prophylaxis, and diagnosis of FNAIT (fetal and neonatal alloimmunethrombocytopenia).

Human platelet alloantigens HPA-1a and HPA-1b are defined by a singlenucleotide mutation resulting in a leucine to proline substitution atposition 33 on the β3 chain of allbβ3 platelet integrin (glycoproteinllbllla). Carriers of a leucine at position 33 of the β-integrin chainare defined as HPA-1a positive, whereas homozygous carriers of a prolineat position 33 of the β-integrin chain are defined as HPA-1a negative(HPA-1bb).

About 2% of Caucasians are homozygous for HPA-1b (P33). Women with thisphenotype may become immunized to HPA-1a in connection with pregnancy,when the foetus has a paternally-inherited HPA-1a allotype.

Mismatch between fetal and maternal HPA-1alloantigens may lead tomaternal immunization with the production of IgG anti-HPA-1a antibodies.These antibodies can traverse the placenta, bind fetal platelets and mayaccelerate platelet destruction causing FNAIT. FNAIT is a seriouscomplication in foetal and neonatal development. Anti-HPA-1a antibodiesaccount for most (85-90%) of FNAIT cases, and are often involved inpost-transfusion purpura (PTP) and in platelet transfusionrefractoriness.

Maternal anti-HPA-1a antibodies produced during a non-compatiblepregnancy can traverse the placenta and cause FNAIT in the fetus of afirst pregnancy (i.e. in the fetus being carried at the time of maternalimmunization). Such fetuses may develop severe thrombocytopenia veryearly during pregnancy. During such a first pregnancy, FNAIT is oftennot detected until birth when the newborn presents with classic symptomsof thrombocytopenia.

As well as affecting a first non-compatible pregnancy, the recurrence ofFNAIT in subsequent non-compatible pregnancies (i.e. pregnancies inwhich a mother who was immunised to the HPA-1alloantigen in connectionwith a first pregnancy is pregnant again with a HPA-1a positive fetus)has been estimated to be more than 80%. Immunisation with the HPA-1aalloantigen may also occur in connection with delivery, which means thata subsequent non-compatible pregnancy may be a risk of FNAIT even if thefirst fetus/newborn was unaffected. Currently, there is no safe andeffective strategy to treat or prevent FNAIT. Furthermore, the conditionis usually not evident until after delivery of a severelythrombocytopenic child. Thus, efficient management of FNAIT will dependon introduction of general screening to identify at-risk pregnancies,and development of prophylaxis and new treatment approaches.

For hemolytic disease of the fetus and newborn (HDFN), a pregnancyrelated disorder caused by antibodies reactive with a fetal red cellalloantigen, an effective antibody-based prophylaxis has been in routineuse for decades. A large prospective screening study in Norway revealedthat HPA-1a immunization can occur in connection with delivery, andtherefore, similar to HDFN, prophylaxis with anti-HPA-1a antibodies maythus prevent FNAIT. Furthermore, experiments employing a murine model ofFNAIT suggested that immunization against HPA-1a can be prevented byadministration of HPA-1a-specific antibodies in connection withdelivery. As a consequence of the above findings, clinical trials areunderway to test the potential of hyperimmune anti-HPA-1a IgG isolatedfrom donor plasma in preventing HPA-1a immunization in connection withpregnancy. Hyperimmune anti-HPA-1a IgG is IgG extracted from womenHPA-1a-alloimmunized in connection with pregnancy.

The inventors believe that an attractive source of anti-HPA-1aantibodies for eventual FNAIT prophylaxis or therapy is recombinantmonoclonal antibodies (mAbs). In contrast to IgG preparations extractedfrom donor plasma, mAbs may be produced in virtually limitless amounts,the specificity and function of mAbs can be characterized in detail, anda therapeutic dose can be determined more accurately providing morereproducibility in treatment. Anti-HPA-1a mAbs would also be of greatvalue as a screening reagent to identify whether women are HPA-1apositive or HPA-1a negative.

The reported human HPA-1a-specific recombinant mAbs have been developedfrom antibody fragments isolated by phage display. It has been suggestedthat antibodies with randomly paired heavy and light chains in vitro(e.g. antibodies prepared by phage display may represent foreignproteins or be autoreactive and therefore are more likely to induceundesirable immune reactions in recipients.

Several mAbs which bind to HPA-1a exist. Two such mAbs were generated inmice by conventional hybridoma technology. One of these, clone LK-4,differentiates HPA-1a from HPA-1b antigens on platelet extracts but notwhen present on intact platelets while a second, SZ21, binds HPA-1a onintact platelets. However, the SZ21 mAb also binds detectably toHPA-1a-negative platelets when used at increasing antibodyconcentrations.

What are needed in the art are new, preferably improved, agents, such asantibodies, that can be used for the treatment, prophylaxis anddiagnosis of FNAIT and for detecting the presence or absence of (i.e.screening for) the presence or absence of the HPA-1a alloantigen in asubject.

The present inventors have identified monoclonal antibodies which bindspecifically to HPA-1a. Using B-cells from a HPA-bb woman who had becomeimmunized in connection with pregnancy with a HPA-1a -positive child,the inventors prepared a clonal B cell line generated byEBV-transformation of memory B-cells and selected single B-cells whichproduced anti-HPA-1a antibodies. The inventors also prepared recombinantversions of these antibodies. The antibodies generated by the inventorshave advantageous properties which make them ideal agents for theabove-mentioned uses,

Thus, in a first aspect, the present invention provides an isolatedantibody that specifically binds to HPA-1a and that comprises at leastone heavy chain variable region that comprises three CDRs and at leastone light chain variable region that comprises three CDRs, wherein saidlight chain variable region comprises:

-   -   (a) a variable light (VL) CDR1 that has the amino acid sequence        of SEQ ID NO:8 or a sequence substantially homologous thereto,    -   (b) a VL CDR2 that has the amino acid sequence of SEQ ID NO:9 or        a sequence substantially homologous thereto, and    -   (c) a VL CDR3 that has the amino acid sequence of SEQ ID NO:10        or a sequence substantially homologous thereto; and

wherein said heavy chain variable region comprises:

-   -   (d) a variable heavy (VH) CDR1 that has the amino acid sequence        of SEQ ID NO:5 or a sequence substantially homologous thereto,    -   (e) a VH CDR2 that has the amino acid sequence of SEQ ID NO:6 or        a sequence substantially homologous thereto, and    -   (f) a VH CDR3 that has the amino acid sequence of SEQ ID NO:7 or        a sequence substantially homologous thereto.

In a preferred embodiment, the invention provides an antibody thatspecifically binds to HPA-1a and that comprises:

-   a VL domain that comprises a VL CDR1 of SEQ ID NO:8, a VL CDR2 of    SEQ ID NO:9, and a VL CDR3 of SEQ ID NO:10, and-   a VH domain that comprises a VH CDR1 of SEQ ID NO:5, a VH CDR2 of    SEQ ID NO:6, and a VH CDR3 of SEQ ID NO:7.

Certain preferred embodiments of the invention provide an antibody thatspecifically binds to HPA-1a comprising a VH domain that comprises theamino acid sequence of SEQ ID NO:3 or a sequence substantiallyhomologous thereto and/or a VL domain that comprises the amino acidsequence of SEQ ID NO:4,or a sequence substantially homologous thereto.

Further preferred embodiments provide an antibody that specificallybinds to HPA-1a comprising a VH domain that comprises the amino acidsequence of SEQ ID NO:3 and a VL domain that comprises the amino acidsequence of SEQ ID NO:4.

Other preferred embodiments of the invention are full length IgG forms(e.g. IgG1 or IgG3) of the antibodies described herein. Thus, apreferred embodiment of the invention provides an antibody that thatspecifically binds to HPA-1a which has a heavy chain that comprises theamino acid sequence of SEQ ID NO:21 or a sequence substantiallyhomologous thereto and/or a light chain that comprises the amino acidsequence of SEQ ID NO:22 or a sequence substantially homologous thereto.In another preferred embodiment the invention provides an antibody thatspecifically binds to HPA-1a which has a heavy chain that comprises theamino acid sequence of SEQ ID NO:25 or a sequence substantiallyhomologous thereto and/or a light chain that comprises the amino acidsequence of SEQ ID NO:26 or a sequence substantially homologous thereto.

In a particularly preferred embodiment, an antibody comprises a heavychain that comprises the amino acid sequence of SEQ ID NO:21and a lightchain that comprises the amino acid sequence of SEQ ID NO:22.

In another particularly preferred embodiment, an antibody comprises aheavy chain that comprises the amino acid sequence of SEQ ID NO:25 and alight chain that comprises the amino acid sequence of SEQ ID NO:26.

The invention is exemplified by the monoclonal antibody D18BL26.4 (seethe Examples section, also referred to herein as “26.4”). The CDRdomains, VH and VL domains and full length IgG chains of the 26.4antibody are shown in Table 1. Antibodies comprising these CDR domains,VH and VL domains, or IgG chains (or sequences substantially homologousthereto) are preferred aspects of the invention. The antibody 26.4,including recombinant versions thereof, represent preferred embodimentsof the invention.

The present invention also provides binding proteins that specificallybind to HPA-1a and that comprise an antibody of the invention.

As used herein, the term “that specifically binds to HPA-1a” in thecontext of antibodies or antibody fragments of the present invention,means antibodies or antigen binding fragments that are capable ofbinding to the alloantigen HPA-1a and which do not cross-react with thealloantigen HPA-1b (i.e. exhibit no significant binding to the HPA-1balloantigen). The 26.4 antibody exemplified herein is an example of anantibody that specifically binds to HPA-1a.

In one embodiment, an antibody of the invention does not cross-reactwith HPA-1b when used at a concentration of 10 μg/ml to 20 μg/ml in theIgG format (e,g. 10 μg/ml or 20 μg/ml), for example when tested againstHPA-1b antigen in a Surface Plasmon Resonance assay (e.g. the assaydescribed in Example 1). Antibodies that are only pseudo specific forHPA-1a are not deemed to specifically bind to HPA-1a in accordance withthe present invention.

Of course, antibody which “binds specifically to HPA-1a” in accordancewith the present invention does not cross-react with other HPA ornon-HPA antigens.

Certain examples of substantially homologous sequences are sequencesthat have at least 70% identity to the amino acid sequences disclosed.

In certain embodiments, the antibodies of the invention that bindspecifically to HPA-1a comprise at least one light chain variable regionthat includes an amino acid sequence region of at least about 70% or75%, more preferably at least about 80%, more preferably at least about85%, more preferably at least about 90% or 95% and most preferably atleast about 97%, 98% or 99% amino acid sequence identity to the aminoacid sequence of SEQ ID NO:4; and/or at least one heavy chain variableregion that includes an amino acid sequence region of at least about 70%or 75%, more preferably at least about 80%, more preferably at leastabout 85%, more preferably at least about 90% or 95% and most preferablyat least about 97%, 98% or 99% amino acid sequence identity to the aminoacid sequence of SEQ ID NO:3.

Other preferred examples of substantially homologous sequences aresequences containing conservative amino acid substitutions of the aminoacid sequences disclosed.

Other preferred examples of substantially homologous sequences aresequences containing 1, 2 or 3, preferably 1 or 2, altered amino acidsin one or more of the CDR regions disclosed. Such alterations might beconserved or non-conserved amino acid substitutions, or a mixturethereof.

In all such embodiments, preferred alterations are conservative aminoacid substitutions.

In a preferred embodiment, the invention provides an isolated antibodythat specifically binds to HPA-1a and that comprises at least one heavychain variable region that comprises three CDRs and at least one lightchain variable region that comprises three CDRs, wherein said lightchain variable region comprises:

-   -   (a) a variable light (VL) CDR1 that has the amino acid sequence        of SEQ ID NO:8 or a sequence substantially homologous thereto,    -   (b) a VL CDR2 that has the amino acid sequence of SEQ ID NO:9 or        a sequence substantially homologous thereto and    -   (c) a VL CDR3 that has the amino acid sequence of SEQ ID NO:10        or a sequence substantially homologous thereto; and

wherein said heavy chain variable region comprises:

-   -   (d) a variable heavy (VH) CDR1 that has the amino acid sequence        of SEQ ID NO:5 or a sequence substantially homologous thereto,    -   (e) a VH CDR2 that has the amino acid sequence of SEQ ID NO:6 or        a sequence substantially homologous thereto, and    -   (f) a VH CDR3 that has the amino acid sequence of SEQ ID NO:7 or        a sequence substantially homologous thereto; and

wherein said substantially homologous sequence is a sequence containing1, 2 or 3 amino acid substitutions compared to the given CDR sequence,or wherein said substantially homologous sequence is a sequencecontaining conservative amino acid substitutions of the given CDRsequence.

In another preferred embodiment, the present invention provides anantibody which specifically binds to HPA-1a , wherein the light chainvariable region has the amino acid sequence of SEQ ID NO:4, or asequence having at least 80% sequence identity thereto and/or whereinthe heavy chain variable region has the amino acid sequence of SEQ IDNO:3, or a sequence having at least 80% sequence identity thereto,

In all embodiments, the antibodies containing substantially homologoussequences retain the ability to specifically bind to HPA-1a andpreferably retain one or more of the other properties described herein,more preferably all of the properties described in relation to the 26.4antibody.

Further examples of substantially homologous amino acid sequences inaccordance with the present invention are described elsewhere herein.

The CDRs of the antibodies of the invention are preferably separated byappropriate framework regions such as those found in naturally occurringantibodies and/or effective engineered antibodies. Thus, the V_(H),V_(L) and individual CDR sequences of the invention are preferablyprovided within or incorporated into an appropriate framework orscaffold to enable antigen binding. Such framework sequences or regionsmay correspond to naturally occurring framework regions, FR1, FR2, FR3and/or FR4, as appropriate to form an appropriate scaffold, or maycorrespond to consensus framework regions, for example identified bycomparing various naturally occurring framework regions. Alternatively,non-antibody scaffolds or frameworks, e.g., T cell receptor frameworkscan be used.

Appropriate sequences that can be used for framework regions are wellknown and documented in the art and any of these may be used. Preferredsequences for framework regions are one or more of the framework regionsmaking up the V_(H) and/or V_(L) domains of the invention, i.e., one ormore of the framework regions of the 26.4 antibody, as disclosed inTable 1, or framework regions substantially homologous thereto, and inparticular framework regions that allow the maintenance of antigenspecificity, for example framework regions that result in substantiallythe same or the same 3D structure of the antibody, In certain preferredembodiments, all four of the variable light chain (SEQ ID NOs:15, 16, 17and 18) and/or variable heavy chain (SEQ ID NOs:11, 12, 13 and 14)framework regions (FR), as appropriate, or FR regions substantiallyhomologous thereto, are found in the antibodies of the invention.

Without wishing to be bound by theory, it is believed that a good systemfor selecting for clinically useful anti-HPA-1a antibodies is to harnessthe selective mechanism in the immune response raised against HPA-1a inHPA-1a negative individuals, i.e. the immune response raised to HPA-1ain HPA-1a negative individuals who have become immunised in connectionwith a non-compatible pregnancy (a pregnancy with a HPA-1a positivefetus). Memory B cells that are selected in such responses should havereceptors that react well to HPA-1a, but not be reactive to theallogeneic antigen HPA-1 b (i.e, “self”). Antibodies selected frommemory B cells of such an individual would thus be expected to be highlyspecific for HPA-1a and not to cross-react with HPA-1b (which onlydiffers from HPA-1a by a single amino acid polymorphism). Furthermore,as an antibody selected by such a system is selected by nature (i.e. bya human immune system) it means that, when used clinically, there shouldbe a reduced risk of anaphylaxis, autoreactivity and/or toxicity, and/orthe antibody should not be rapidly removed from the circulation, ascompared to, for example, an antibody selected in vitro from a library(e.g. by phage display). The antibodies of the present invention arebased on the antibody 26.4, which was selected in such a manner. The26.4 antibody was derived from a single B-cell of a HPA-1a negativewoman who was HPA-1a alloimmunised in connection with pregnancy.

Thus, preferably the antibodies of the present invention have a low riskof causing anaphylaxis and/or toxicity when used clinically. Preferably,the antibodies of the invention are not autoreactive. In certainembodiments, the antibodies of the invention are not rapidly clearedfrom the circulation.

As described above, antibodies of the invention bind specifically toHPA-1a. Preferably, the antibodies bind to HPA-1a on intact platelets.Assays to ascertain whether antibodies bind to HPA-1a on intact HPA-1apositive platelets include, but are not limited to flow cytometry (e.g.whole blood flow cytometry) or the MAIPA assay (monoclonal antibodyimmobilization of platelet antigens assay). Suitable flow cytometry andMAIPA assays are described in the Examples. in certain embodiments, theantibodies of the invention are capable of binding to purified forms ofHPA-1a or HPA-1a bearing proteins. As described above, the HPA-1aantigen is present in allbβ3 platelet integrin (glycoprotein llbllla).Preferably, antibodies of the present invention are capable of bindingto purified αllbβ3 platelet integrin from HPA-1a positive individuals.Methods for purifying αllbβ3 platelet integrin are known in the art, asare methods for determining whether an antibody is able to bind to apurified protein. For example, Example 1 describes a method forpurifying (isolating) αllbβ3 platelet integrin from platelets. Example1also describes how Surface Plasmon Resonance can be used to analyse thebinding of purified forms of HPA-1a or HPA-1a bearing proteins.Preferred antibodies remain at least 50% bound to a purified andimmobilised αllbβ3 platelet integrin from HPA-1a positive individuals atthe end of the dissociation period in a Surface Plasmon Resonance assay.Preferably, at least 60%, at least 70%, at least 80% or at least 90% ofthe antibody remains bound at the end of the dissociation period in aSurface Plasmon Resonance assay. For example, about 50% to about 80% ofthe antibody remains bound. A preferred association period in such aSurface Plasmon Resonance assay is 120 seconds. A preferred dissociationperiod in such a Surface Plasmon Resonance assay is 120 seconds. Aparticularly preferred Surface Plasmon Resonance assay is described inExample 1.

αVβ3 integrin is another β3 integrin-containing heterodimer (vitronectinreceptor), αVβ3 is expressed on fetal trophoblast cells. αVβ3 integrinon fetal trophoblast cells obtained from a HPA-1a positive individual(e.g. a HPA-1a homozygous individual) or purified from such anindividual contains the HPA-1a antigen. Thus, antibodies which bindspecifically to αVβ3 integrin from HPA-1a positive individuals areconsidered antibodies that specifically bind to HPA-1a in accordancewith the present invention.

Fetal trophoblasts, which line the maternal-fetal interphase, areconstantly released into the maternal circulation throughout pregnancy.Thus, HPA-1a positive fetal trophoblasts represent a source of HPA-1afor allommunization of a woman during a non-compatible pregnancy, i.e. apregnancy where the mother is HPA-1a negative and the fetus is HPA-1apositive. It is known that some women become immunized to HPA-1a at anearly time point in pregnancy, when immunization with fetal platelets isunlikely due to the developmental stage of fetal blood cells. In suchcases αVβ3 integrin containing the HPA-1a antigen is the likelyimmunogen. Thus, antibodies of the present invention which are capableof binding to αVβ3 integrin containing the HPA-1a antigen are preferred.

In certain embodiments, the antibodies of the invention are capable ofbinding to the HPA-1a antigen on intact fetal trophoblasts.

In certain embodiments, the antibodies of the invention are capable ofbinding to purified αVβ3 integrin from HPA-1a positive individuals.Methods for purifying αVβ3 integrin are known in the art, as are methodsfor determining whether an antibody is able to bind to a purifiedprotein. For example, Example 1 describes a method for purifying(isolating) αVβ3 integrin from human placenta. Example 1also describeshow Surface Plasmon Resonance can be used to analyse the binding ofpurified forms of HPA-1a or HPA-1a bearing proteins. Preferredantibodies remain at least 35% bound to a purified and immobilised αVβ3integrin from HPA-1a positive individuals at the end of the dissociationperiod in a Surface Plasmon Resonance assay. Preferably, at least 40%,at least 45%, at least 50%, at least 55%, at least 60% or at least 65%of the antibody remains bound at the end of the dissociation period in aSurface Plasmon Resonance assay. For example, 35% to 70% of the antibodyremains bound. A preferred association period in such a Surface PlasmonResonance assay is 120 seconds. A preferred dissociation period in sucha Surface Plasmon Resonance assay is 120 seconds. Suitable antigen(ligand) densities on the chip used in Surface Plasmon Resonance areknown in the art and can readily be established (e.g. those of Example1). A particularly preferred Surface Plasmon Resonance assay isdescribed in Example 1.

Preferably, in Surface Plasmon Resonance experiments, antibodies of thepresent invention dissociate from purified and immobilised αVβ3 integrinfrom HPA-1a positive individuals slower than the antibody B2G1 (GriffinH, Ouwehand W., Blood. 1995; 86(12):4430-6). For example, antibodies ofthe present invention dissociate about 10%, about 20%, about 30%, about40%, about 50%, about 60%, about 70%, about 80%, about 90% or about 100%slower than the antibody B2G1 (e.g. about 50% to about 100% slower).

Preferably, in Surface Plasmon Resonance experiments, antibodies of theinvention have a higher binding response for αVβ3 integrin from HPA-1apositive individuals than the antibody B2G1. Preferably, the bindingresponse is at least 10%, at least 20%, at least 30%, at least 40%, atleast 50%, at least 60% or at least 70% higher than for the antibodyB2G1 (e.g. about 10% to about 70% higher). Binding response is theresponse units (RU) value at the end of the association phase.

The amino acid sequences of the heavy chain variable region and lightchain variable region of B2G1are as follows:

Heavy chain variable region of B2G1 (SEQ ID NO: 27)QVQLVQSGAEVKRPGAAVKVSCKASGYRFTGHYMBNVRQAPGQGLEWMGWINPNSGGTSYAQKFQGRVIMTRDTSISTAYMEMTRLRYD2TAVnCAAGGL GGYYYYAMNIWCGITVTVSSLight chain variable region of B2G1 (SEQ ID NO: 28) QSALTUASVSGSPGQSITISCTGISSDVGGYNYVSWYQQHPGKAPKLMIYEVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTWVF GGGTKLTVL

In some embodiments, antibodies of the invention have the ability toinhibit the binding of the anti-HPA-1a antibody SZ21 to αVβ3 integrin(HPA-1aa genotyped). Inhibition of binding does not necessarily mean acomplete block on binding, inhibition includes a significant ormeasurable reduction in binding. Preferably, the ability to inhibit thebinding of the anti-HPA-1a antibody SZ21 to αVβ3 integrin is adose-dependent ability, i.e. as the concentration of an antibody of theinvention increases, the inhibition of binding of the anti-HPA-1aantibody SZ21 to αVβ3 integrin increases. Suitable assays for assessingthe ability of a given antibody to inhibit the binding of theanti-HPA-1a antibody SZ21 to αVβ3 integrin are known in the art. In suchassays the αVβ3 integrin may be from a cell lysate from a trophoblastcell line (e.g. the TCL-1 cell line, Lewis M P, et al., (1996), Placenta17: 137-46). A particularly suitable assay is a flow cytometric antibodybinding-inhibition assay, for example the flow cytometric antibodybinding inhibition assay described in Example 1.

In preferred embodiments, antibodies of the present invention have anincreased ability to inhibit the binding of the anti-HPA-1a antibodySZ21 to αVβ3 integrin (HPA-1aa genotyped) compared to the ability of theantibody B2G1 to inhibit the binding of the anti-HPA-1a antibody SZ21 toαVβ3 integrin (HPA-1aa genotyped). Put another way, in some embodimentsantibodies of the present invention are more efficient than the antibodyB2G1at inhibiting the binding of the anti-HPA-1a antibody SZ21 to αVβ3integrin (HPA-1aa genotyped). For example, in some embodiments, whenused at an amount of 12.5 ng-200 ng (e.g. 12.5 ng, 25 ng, 50 ng, 100 ngor 200 ng) antibodies of the present invention have an increased abilityto inhibit the binding of the anti-HPA-1a antibody SZ21 to αVβ3 integrin(HPA-1aa genotyped) compared to the ability of the antibody B2G1 (usedat the same amount/concentrations) to inhibit the binding of theanti-HPA-1a antibody SZ21 to αVβ3 integrin (HPA-1aa genotyped).Typically, the antibodies are used in a fixed volume of 200 μl so theabove-mentioned amounts of 12.5 ng, 25 ng, 50 ng, 100 ng and 200 ngequate to concentrations of 62.5 ng/ml, 125 ng/ml, 250 ng/ml, 500 ng/mland 1000 ng/ml, respectively. This increased ability is significant andpreferred antibodies inhibit as well as antibody 26.4, e.g. as shown inFIG. 5F). Preferred antibodies are at least 20%, preferably at least30%, more preferably at least 40 or 50% more effective (at any of theaforementioned concentrations) at inhibiting binding of SZ21 to αVβ3integrin than B2G1 is.

The amino acid sequences of the heavy chain variable region and lightchain variable region of SZ21are as follows:

Heavy chain variable region of SZ21 (Genebank Accession Number AF354053)(SEQ ID NO: 29) LQESGPELVNPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWIGLINPYHGGSSYNQKFKGKATLTVDKSSSTAVMELLSLTSEDSAVYFCARRDANY VFFFDYWGQGTTVTLight chain variable region of SZ21 (Genebank Accession Number AF354054)(SEQ ID NO: 30) ELTQSPALMSASPGEKVTMTCSASSGVSYIHWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSKPPTFGGGTK LE

In preferred embodiments, the antibodies of the invention are capable ofinducing phagocytosis of HPA-1a positive platelets. Without wishing tobe bound by theory, it is believed that the antibodies act by binding toHPA-1a on platelets and sensitizing/opsonizing the bound platelets fordestruction by phagocytes (e.g. monocytes). Thus, the ability to inducephagocytosis of HPA-1a positive platelets is believed to be particularlyimportant in the context of FNAIT prophylaxis. Preferably, antibodies ofthe invention induce phagocytosis of HPA-1a positive platelets in aconcentration dependent manner, with increased phagocytosis beingobserved as the antibody concentration used increases. In certainembodiments, the antibodies of the invention are capable of inducingphagocytosis when used at a concentration of at least 0.05 μg/ml, forexample at a concentration in the range of 0.05 μg/ml to 50 μg/ml,preferably at a concentration of about 0.1 μg/ml to about 10 μg/ml. Inpreferred embodiments, antibodies induce at least 20% plateletphagocytosis, for example at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90% phagocytosis or 100%phagocytosis (e.g. about 30% to about 90% phagocytosis). For example, incertain embodiments, antibodies in accordance with the present inventionare capable of inducing about 90% HPA-1a homozygous plateletphagocytosis when used at a concentration of 10 μug/ml. Preferably, theantibodies of the invention do not induce phagocytosis of HPA-1anegative platelets. Methods for assessing platelet phagocytosis areknown in the art and a suitable assay is described in Example 1. The %phagocytosis value may be the % of monocytes having internalizedplatelets, e.g. in an assay as described in Example 1. The assaydescribed in Example 1 is a preferred assay for assessing the ability ofantibodies of the present invention to induce phagocytosis.

Preferably the antibodies of the invention do not inhibit aggregation ofHPA-1bb platelets (e.g. less than 10% inhibition at an antibodyconcentration of 12 μg/ml). Preferably the antibodies of the inventiondo not greatly inhibit aggregation of HPA-1ab platelets (e.g. no morethan 30%, preferably no more than 20% inhibition at an antibodyconcentration of 12 μg/ml). This lack of significant inhibitory activitymeans the antibodies will not impede the function of maternal or fetalplatelets. The antibodies of the invention will, in addition, preferablynot have an activatory effect on HPA-1a positive platelets (e.g. at anantibody concentration of 12 μg/ml).

Methods for assessing an effect on platelet aggregation are known in theart and a suitable assay is described in Example 1. The assay describedin Example 1 is a preferred assay for assessing the ability ofantibodies of the present invention to inhibit platelet aggregation.

In some embodiments, the antibodies of the invention are capable ofinhibiting the binding of maternal polyclonal anti HPA 1a IgG to HPA-1ahomozygous platelets. In one such embodiment the antibody is preferablya F(ab′)₂ fragment of the 26.4 antibody. Preferably, the inhibition isat least 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, or 100%. For example, theinhibition may be 65%-100%. Such inhibition can be assessed using aMAIPA assay, for example as described in Example 3 herein.

As used throughout the entire application, the terms “a” and “an” areused in the sense that they mean “at least one”, “at least a first”,“one or more” or “a plurality” of the referenced components or steps,except in instances wherein an upper limit is thereafter specificallystated. Therefore, an “antibody”, as used herein, means “at least afirst antibody”. The operable limits and parameters of combinations, aswith the amounts of any single agent, will be known to those of ordinaryskill in the art in light of the present disclosure.

Nucleic acid molecules comprising nucleotide sequences that encode theantibodies of the present invention as defined herein or parts orfragments thereof, or nucleic acid molecules substantially homologousthereto, form yet further aspects of the invention. A preferred nucleicacid is a nucleic acid encoding a heavy chain of an antibody (e.g.,those encoding SEQ ID NOs:21and 25, such as SEQ ID NO:19 and SEQ IDNO:23, respectively) or those encoding a light chain of an antibody(e.g., those encoding SEQ ID NOs:22 and 26, such as SEQ ID NOs:20 and24). Other preferred nucleic acid molecules are those encoding a VHregion of an antibody of the present invention (e.g., those encoding SEQID NO:3, such as SEQ ID NO:1). Other preferred nucleic acid moleculesare those encoding a VL region of an antibody of the present invention(e.g., those encoding SEQ ID NO:4, such as SEQ ID NO:2).

The term “substantially homologous” as used herein in connection with anamino acid or nucleic acid sequence includes sequences having at least70% or 75%, preferably at least 80%, and even more preferably at least85%, 90%, 95%, 96%, 97%, 98% or 99%, sequence identity to the amino acidor nucleic acid sequence disclosed. Substantially homologous sequencesof the invention thus include single or multiple base or amino acidalterations (additions, substitutions, insertions or deletions) to thesequences of the invention. At the amino acid level preferredsubstantially homologous sequences contain up to 5, e.g. only 1, 2, 3, 4or 5, preferably 1, 2 or 3, more preferably 1 or 2, altered amino acids,in one or more of the framework regions and/or one or more of the CDRsmaking up the sequences of the invention. Said alterations can be withconservative or non-conservative amino acids. Preferably saidalterations are conservative amino acid substitutions.

The term “substantially homologous” also includes modifications orchemical equivalents of the amino acid and nucleotide sequences of thepresent invention that perform substantially the same function as theproteins or nucleic acid molecules of the invention in substantially thesame way. For example, any substantially homologous antibody shouldretain the ability to bind to HPA-1a as described above. Preferably, anysubstantially homologous antibody should retain one or more of thefunctional capabilities of the starting antibody.

Preferably, any substantially homologous antibody should retain theability to specifically bind to the same epitope of HPA-1a as recognizedby the antibody in question, for example, the same epitope recognized bythe CDR domains of the invention or the VH and VL domains of theinvention as described herein. Binding to the same epitope/antigen canbe readily tested by methods well known and described in the art, e.g.,using binding assays, e.g., a competition assay. Retention of otherfunctional properties can also readily be tested by methods well knownand described in the art.

Thus, a person skilled in the art will appreciate that binding assayscan be used to test whether “substantially homologous” antibodies havethe same binding specificities as the antibodies and antibody fragmentsof the invention, for example, binding assays such as ELISA assays orBIAcore assays can readily be used to establish whether such“substantially homologous” antibodies can bind to HPA-1a. As outlinedbelow, a competition binding assay can be used to test whether“substantially homologous” antibodies retain the ability to specificallybind to substantially the same epitope of HPA-1a as recognized by theantibodies of the invention (e.g. 26.4), or have the ability to competewith one or more of the various antibodies of the invention (e.g. 26.4).The method described below is only one example of a suitable competitionassay. The skilled person will be aware of other suitable methods andvariations.

An exemplary competition assay involves assessing the binding of variouseffective concentrations of an antibody of the invention to HPA-1a inthe presence of varying concentrations of a test antibody (e.g., asubstantially homologous antibody). The amount of inhibition of bindinginduced by the test antibody can then be assessed. A test antibody thatshows increased competition with an antibody of the invention atincreasing concentrations (i.e., increasing concentrations of the testantibody result in a corresponding reduction in the amount of antibodyof the invention binding to HPA-1a ) is evidence of binding tosubstantially the same epitope. Preferably, the test antibodysignificantly reduces the amount of antibody of the invention that bindsto HPA-1a. Preferably, the test antibody reduces the amount of antibodyof the invention that binds to HPA-1a by at least about 95%. ELISA andflow cytometry assays are appropriate for assessing inhibition ofbinding in such a competition assay but other suitable techniques wouldbe well known to a person skilled in the art.

Substantially homologous sequences of proteins of the invention include,without limitation, conservative amino acid substitutions, or forexample alterations that do not affect the VH, VL or CDR domains of theantibodies, e.g., antibodies where tag sequences or other components areadded that do not contribute to the binding of antigen, or alterationsto convert one type or format of antibody molecule or fragment toanother type or format of antibody molecule or fragment (e.g.,conversion from Fab to scFv or vice versa), or the conversion of anantibody molecule to a particular class or subclass of antibody molecule(e.g., the conversion of an antibody molecule to IgG or a subclassthereof, e.g., IgG1 or IgG3).

A “conservative amino acid substitution”, as used herein, is one inwhich the amino acid residue is replaced with another amino acid residuehaving a similar side chain. Families of amino acid residues havingsimilar side chains have been defined in the art, including basic sidechains (e.g., lysine, arginine, histidine), acidic side chains (e.g.,aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., glycine, cysteine, alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine),

Homology may be assessed by any convenient method. However, fordetermining the degree of homology between sequences, computer programsthat make multiple alignments of sequences are useful, for instanceClustal W (Thompson, Higgins, Gibson, Nucleic Acids Res., 22:4673-4680,1994). If desired, the Clustal W algorithm can be used together withBLOSUM 62 scoring matrix (Henikoff and Henikoff, Proc. Natl. Acad, Sci.USA, 89:10915-10919, 1992) and a gap opening penalty of 10 and gapextension penalty of 0.1, so that the highest order match is obtainedbetween two sequences wherein at least 50% of the total length of one ofthe sequences is involved in the alignment. Other methods that may beused to align sequences are the alignment method of Needleman and Wunsch(Needleman and Wunsch, J. Mol. Biol., 48:443, 1970) as revised by Smithand Waterman (Smith and Waterman, Adv. Appl, Math., 2:482, 1981) so thatthe highest order match is obtained between the two sequences and thenumber of identical amino acids is determined between the two sequences.Other methods to calculate the percentage identity between two aminoacid sequences are generally art recognized and include, for example,those described by Carillo and Lipton (Carillo and Lipton, SIAM J.Applied Math., 48:1073, 1988) and those described in ComputationalMolecular Biology, Lesk, e.d. Oxford University Press, New York, 1988,Biocomputing; Informatics and Genomics Projects.

Generally, computer programs will be employed for such calculations.Programs that compare and align pairs of sequences, like ALIGN (Myersand Miller, CABIOS, 4:11-17, 1988), FASTA (Pearson and Lipman. Proc.Natl. Acad. Sci. USA, 85:2444-2448, 1988; Pearson, Methods inEnzymology, 183:63-98, 1990) and gapped BLAST (Altschul et al., NucleicAcids Res., 25;3389-3402, 1997), BLASTP, BLASTN, or GCG (Devereux,Haeberli, Smithies, Nucleic Acids Res., 12:387, 1984) are also usefulfor this purpose. Furthermore, the Dali server at the EuropeanBioinformatics institute offers structure-based alignments of proteinsequences (Holm, Trends in Biochemical Sciences, 20;478-480, 1995; Holm,J. Mol. Biol., 233:123-38, 1993; Holm, Nucleic Acid Res., 26:316-9,1998).

By way of providing a reference point, sequences according to thepresent invention having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or99% homology, sequence identity etc. may be determined using the ALIGNprogram with default parameters (for instance available on Internet atthe GENESTREAM network server, IGH, Montpellier, France).

In the following descriptions of the compositions, immunoconjugates,pharmaceuticals, combinations, cocktails, kits, first and second medicaluses and all methods in accordance with this invention, the terms“antibody” and “immunoconjugate”, or an antigen-binding region orfragment thereof, unless otherwise specifically stated or made clearfrom the scientific terminology, refer to a range of anti-HPA-1aantibodies as well as to the specific 26.4 antibody.

The terms “antibody” and “immunoglobulin”, as used herein, refer broadlyto any immunological binding agent that comprises an antigen bindingdomain (e.g. a human antigen binding domain), including polyclonal andmonoclonal antibodies. Depending on the type of constant domain in theheavy chains, whole antibodies are assigned to one of five majorclasses: IgA, IgD, IgE, IgG, and IgM and the antibodies of the inventionmay be in any one of these classes. Several of these are further dividedinto subclasses or isotypes, such as IgG1, IgG2, IgG3, IgG4, and thelike. The heavy-chain constant domains that correspond to the differenceclasses of immunoglobulins are termed α, δ, ε, γ and μ, respectively.The subunit structures and three-dimensional configurations of differentclasses of immunoglobulins are well known.

Generally, where whole antibodies rather than antigen binding regionsare used in the invention, IgG and/or IgM are preferred because they arethe most common antibodies in the physiological situation and becausethey are most easily made in a laboratory setting. IgG1and IgG3antibodies are particularly preferred.

The “light chains” of mammalian antibodies are assigned to one of twoclearly distinct types: kappa (κ) and lambda (λ), based on the aminoacid sequences of their constant domains and some amino acids in theframework regions of their variable domains.

As will be understood by those in the art, the immunological bindingreagents encompassed by the term “antibody” extend to all humanantibodies and antigen binding fragments thereof, including wholeantibodies, dimeric, trimeric and multimeric antibodies; bispecificantibodies; chimeric antibodies; recombinant and engineered antibodies,and fragments thereof.

The term “antibody” is thus used to refer to any antibody-like moleculethat has an antigen binding region, and this term includes antibodyfragments that comprise an antigen binding domain such as Fab′, Fab,F(ab′)₂, single domain antibodies (DABS), TandAbs dimer, Fv, scFv(single chain Fv), dsFv, ds-scFv, Fd, linear antibodies, minibodies,diabodies, bispecific antibody fragments, bibody, tribody (scFv-Fabfusions, bispecific or trispecific, respectively); sc-diabody;kappa(lamda) bodies (scFv-CL fusions); BiTE (Bispecific T-cell Engager,scFv-scFv tandems to attract T cells); DVD-g (dual variable domainantibody, bispecific format); SIP (small immunoprotein, a kind ofminibody); SMIP (“small modular immunopharmaceutical” scFv-Fc dimer;DART (ds-stabilized diabody “Dual Affinity ReTargeting”); small antibodymimetics comprising one or more CDRs and the like. In one preferredembodiment the antibody fragment is a F(ab′)₂ fragment.

The techniques for preparing and using various antibody-based constructsand fragments are well known in the art. Diabodies, in particular, arefurther described in EP 404 097 and WO 93/11161; whereas linearantibodies are further described in the art.

The term “heavy chain complementarity determining region” (“heavy chainCDR”) as used herein refers to regions of hypervariability within theheavy chain variable region (V_(H) domain) of an antibody molecule. Theheavy chain variable region has three CDRs termed heavy chain CDR1,heavy chain CDR2 and heavy chain CDR3 from the amino terminus to carboxyterminus. The heavy chain variable region also has four frameworkregions (FR1 , FR2, FR3 and FR4 from the amino terminus to carboxyterminus). These framework regions separate the CDRs.

The term “heavy chain variable region” (V_(H) domain) as used hereinrefers to the variable region of a heavy chain of an antibody molecule.

The term “light chain complementarity determining region” (“light chainCDR”) as used herein refers to regions of hypervariability within thelight chain variable region (V_(L) domain) of an antibody molecule.Light chain variable regions have three CDRs termed light chain CDR1,light chain CDR2 and light chain CDR3 from the amino terminus to thecarboxy terminus. The light chain variable region also has fourframework regions (FR1, FR2, FR3 and FR4 from the amino terminus tocarboxy terminus). These framework regions separate the CDRs.

The term “light chain variable region” (V_(L) domain) as used hereinrefers to the variable region of a light chain of an antibody molecule.

Antibodies can be fragmented using conventional techniques. For example,F(ab′)₂ fragments can be generated by treating the antibody with pepsin.The resulting F(ab′)₂ fragment can be treated to reduce disulfidebridges to produce Fab′ fragments. Papain digestion can lead to theformation of Fab fragments, Fab, Fab′ and F(ab′)₂, scFv, Fv, dsFv, Fd,dAbs, TandAbs, ds-scFv, dimers, minibodies, diabodies, bispecificantibody fragments and other fragments can also be synthesized byrecombinant techniques or can be chemically synthesized. Techniques forproducing antibody fragments are well known and described in the art.

In certain embodiments, the antibody or antibody fragment of the presentinvention comprises all or a portion of a heavy chain constant region,such as an IgG1, IgG2, IgG3, lgG4, IgA1, lgA2, IgE, or IgD constantregion. Preferably, the heavy chain constant region is an IgG1 or IgG3heavy chain constant region, or a portion thereof. Furthermore, theantibody or antibody fragment can comprise all or a portion of a kappalight chain constant region or a lambda light chain constant region, ora portion thereof. All or part of such constant regions may be producednaturally or may be wholly or partially synthetic. Appropriate sequencesfor such constant regions are well known and documented in the art. Whena full complement of constant regions from the heavy and light chainsare included in the antibodies of the invention, such antibodies aretypically referred to herein as “full length” antibodies or “whole”antibodies.

The antibodies or antibody fragments can be produced naturally or can bewholly or partially synthetically produced. Thus the antibody may befrom any appropriate source, for example recombinant sources and/orproduced in transgenic animals or transgenic plants, or in eggs usingthe IgY technology. Thus, the antibody molecules can be produced invitro or in vivo.

Preferably, the antibody or antibody fragment comprises an antibodylight chain variable region (V_(L)) that comprises three CDR domains andan antibody heavy chain variable region (V_(H)) that comprises three CDRdomains. Said VL and VH generally form the antigen binding site.

An “Fv” fragment is the minimum antibody fragment that contains acomplete antigen-recognition and binding site. This region has a dimerof one heavy chain and one light chain variable domain in tight,non-covalent association. It is in this configuration that the threehypervariable regions (CDRs) of each variable domain interact to definean antigen-binding site on the surface of the V_(H)-V_(L) dimer.Collectively, the six hypervariable regions (CDRs) conferantigen-binding specificity to the antibody.

However, it is well documented in the art that the presence of threeCDRs from the light chain variable domain and three CDRs from the heavychain variable domain of an antibody is not necessary for antigenbinding. Thus, constructs smaller than the above classical antibodyfragment are known to be effective.

For example, camelid antibodies have an extensive antigen bindingrepertoire but are devoid of light chains. Also, results with singledomain antibodies comprising VH domains alone or VL domains alone showthat these domains can bind to antigen with acceptably high affinities.Thus, three CDRs can effectively bind antigen.

Thus, although preferred antibodies of the invention might comprise sixCDR regions (three from a light chain and three from a heavy chain),antibodies with fewer than six CDR regions (e.g. 3 CDR regions) areencompassed by the invention. Antibodies with CDRs from only the heavychain or light chain are also contemplated.

Preferred light chain CDR regions for use in conjunction with thespecified heavy chain CDR regions are described elsewhere herein,However, other light chain variable regions that comprise three CDRs foruse in conjunction with the heavy chain variable regions of theinvention are also contemplated. Appropriate light chain variableregions which can be used in combination with the heavy chain variableregions of the invention and which give rise to an antibody which bindsHPA-1a can be readily identified by a person skilled in the art.

For example, a heavy chain variable region of the invention can becombined with a single light chain variable region or a repertoire oflight chain variable regions and the resulting antibodies tested forbinding to HPA-1a.

If desired, similar methods could be used to identify alternative heavychain variable regions for use in combination with preferred light chainvariable regions of the invention.

Thus, another aspect of the invention provides an isolated antibody thatspecifically binds to HPA-1a and that comprises at least one heavy chainvariable region that comprises three CDRs and at least one light chainvariable region that comprises three CDRs, wherein said light chainvariable region comprises:

-   -   (a) a variable light (VL) CDR1 that has the amino acid sequence        of SEQ ID NO:8 or a sequence substantially homologous thereto,    -   (b) a VL CDR2 that has the amino acid sequence of SEQ ID NO:9 or        a sequence substantially homologous thereto, and    -   (c) a VL CDR3 that has the amino acid sequence of SEQ ID NO:10        or a sequence substantially homologous thereto.

Substantially homologous sequences are defined elsewhere herein. Incertain embodiments, the substantially homologous sequence is a sequencecontaining 1, 2 or 3 amino acid substitutions compared to the given CDRsequence, or said substantially homologous sequence is a sequencecontaining conservative amino acid substitutions of the given CDRsequence. Other features and properties of other aspects of theinvention apply, mutates mutandis, to this aspect of the invention.

In certain embodiments, the antibody comprises a VL domain thatcomprises the amino acid sequence of SEQ ID NO:4, or a sequencesubstantially homologous thereto (e.g. a sequence having at least 80%sequence identity thereto). In preferred embodiments, the VL domaincomprises the amino acid sequence of SEQ ID NO:4.

In another aspect, the invention provides an isolated antibody thatspecifically binds to HPA-1a and that comprises at least one heavy chainvariable region that comprises three CDRs and at least one light chainvariable region that comprises three CDRs, wherein said heavy chainvariable region comprises:

-   -   (a) a variable heavy (VH) CDR1 that has the amino acid sequence        of SEQ ID NO:5 or a sequence substantially homologous thereto,    -   (b) a VH CDR2 that has the amino acid sequence of SEQ ID NO:6 or        a sequence substantially homologous thereto, and    -   (c) a VH CDR3 that has the amino acid sequence of SEQ ID NO:7 or        a sequence substantially homologous thereto.

Substantially homologous sequences are defined elsewhere herein. Incertain embodiments, the substantially homologous sequence is a sequencecontaining 1, 2 or 3 amino acid substitutions compared to the given CDRsequence, or said substantially homologous sequence is a sequencecontaining conservative amino acid substitutions of the given CDRsequence. Other features and properties of other aspects of theinvention apply, mutatis mutandis, to this aspect of the invention.

In certain embodiments, the antibody comprises a VH domain thatcomprises the amino acid sequence of SEQ ID NO:3, or a sequencesubstantially homologous thereto (e.g. a sequence having at least 80%sequence identity thereto). In preferred embodiments, the VH domaincomprises the amino acid sequence of SEQ ID NO:3.

A yet further aspect of the invention provides an antibody, preferablyan isolated antibody, more preferably a human antibody, whichspecifically binds to HPA-1a and which has the ability to compete with(i.e. bind to the same or substantially the same epitope as) the 26.4antibody (i.e. an antibody comprising the VL of SEQ ID NO:4 and the VHof SEQ ID NO:3) as described herein, or the ability to compete with anantibody comprising the same CDRs as 26.4, i.e. an antibody comprisingVL CDR sequences of SEQ ID NOs: 8, 9 and 10 and VH CDR sequences of SEQID NOs: 5, 6 and 7, for binding to HPA-1a. Other features and propertiesof other aspects of the invention apply, mutatis mutandis, to thisaspect of the invention.

Binding to the same epitope/antigen can be readily tested by methodswell known and described in the art, e.g. using binding assays such as acompetitive inhibition assay. Thus, a person skilled in the art willappreciate that binding assays can be used to identify other antibodiesand antibody fragments with the same binding specificities as theantibodies and antibody fragments of the invention. Suitable bindingassays are discussed elsewhere herein.

in some embodiments, an antibody of the invention is a Type IIanti-HPA-1a antibody. Thus, in some embodiments, an antibody of thepresent invention binds to an epitope on β₃ integrin that is not solelydefined by the PSI (piexin/semaphorin/integrin) domain of β₃ intedrin.In some embodiments, the epitope on β₃ integrin to which antibodies ofthe invention bind includes residues of the PSI(plexin/semaphorin/integrin) domain and, in addition, includes residuesof the hybrid and/or of an epidermal growth factor (EGF) domain(s) of β₃integrin. In some embodiments, the epitope on p, integrin to whichantibodies of the invention bind includes residues of the hybrid and/orof an epidermal growth factor (EGF) domain(s) of β₃ integrin. A suitableassay for identifying domains on β₃ integrin which are bound by anantibody is described in Example 4.

Preferably, the above described abilities and properties are observed ata measurable or significant level and more preferably at a statisticallysignificant level, when compared to appropriate control levels.Appropriate significance levels are discussed elsewhere herein. Morepreferably, one or more of the above described abilities and propertiesare observed at a level which is measurably better, or more preferablysignificantly better, when compared to the abilities observed for priorart antibodies.

In any statistical analysis referred to herein, preferably thestatistically significant difference over a relevant control has aprobability value of <0.1 preferably <005, more preferably <0.01.Appropriate methods of determining statistical significance are wellknown and documented in the art and any of these may be used.

In other preferred embodiments, second generation antibodies areprovided that have enhanced or superior properties in comparison to anoriginal anti-HPA-1a antibody, such as 26.4.

Comparisons to identify effective second generation antibodies arereadily conducted and quantified, e.g., using one or more of the variousassays described in detail herein or in the art. Second generationantibodies that have an enhanced biological property or activity of atleast about 2-fold, 5-fold, 10-fold, 20-fold, and preferably, at leastabout 50-fold, in comparison to the anti-HPA-1a antibodies of thepresent invention, as exemplified by the 26.4 antibody, are encompassedby the present invention.

The antibody, binding protein and nucleic acid molecules of theinvention are generally “isolated” or “purified” molecules insofar asthey are distinguished from any such components that may be present insitu within a human or animal body or a tissue sample derived from ahuman or animal body. The sequences may, however, correspond to or besubstantially homologous to sequences as found in a human or animalbody. Thus, the term “isolated” or “purified” as used herein inreference to nucleic acid molecules or sequences and proteins orpolypeptides, e.g., antibodies, refers to such molecules when isolatedfrom, purified from, or substantially free of their natural environment,e.g., isolated from or purified from the human or animal body (if indeedthey occur naturally), or refers to such molecules when produced by atechnical process, i.e., includes recombinant and synthetically producedmolecules.

Thus, when used in connection with a protein or polypeptide moleculesuch as light chain CDRs 1, 2 and 3, heavy chain CDRs 1, 2 and 3, lightchain variable regions, heavy chain variable regions, and bindingproteins or antibodies of the invention, including full lengthantibodies, the term “isolated” or “purified” typically refers to aprotein substantially free of cellular material or other proteins fromthe source from which it is derived. In some embodiments, particularlywhere the protein is to be administered to humans or animals, suchisolated or purified proteins are substantially free of culture mediumwhen produced by recombinant techniques, or chemical precursors or otherchemicals when chemically synthesized.

The term “nucleic acid sequence” or “nucleic acid molecule” as usedherein refers to a sequence of nucleoside or nucleotide monomerscomposed of naturally occurring bases, sugars and intersugar (backbone)linkages. The term also includes modified or substituted sequencescomprising non-naturally occurring monomers or portions thereof. Thenucleic acid sequences of the present invention may be deoxyribonucleicacid sequences (DNA) or ribonucleic acid sequences (RNA) and may includenaturally occurring bases including adenine, guanine, cytosine,thymidine and uracil. The sequences may also contain modified bases.Examples of such modified bases include aza and deaza adenine, guanine,cytosine, thymidine and uracil; and xanthine and hypoxanthine. Thenucleic acid molecules may be double stranded or single stranded. Thenucleic acid molecules may be wholly or partially synthetic orrecombinant.

In preferred embodiments the antibodies of the invention are humanantibodies, more preferably fully human antibodies.

The term “human” as used herein in connection with antibody moleculesand binding proteins first refers to antibodies and binding proteinshaving variable regions (e.g., V_(H), V_(L), CDR or FR regions) and,optionally, constant antibody regions, isolated or derived from a humanrepertoire or derived from or corresponding to sequences found inhumans, e.g., in the human germline or somatic cells. The 26.4 antibodyis an example of such a human antibody molecule wherein the variableregions correspond to sequences found in a human.

The term “fragment” as used herein refers to fragments of biologicalrelevance, e.g., fragments that contribute to antigen binding, e.g.,form part of the antigen binding site, and/or contribute to theinhibition or reduction in function of the HPA-1a antigen. Certainpreferred fragments comprise a heavy chain variable region (V_(H)domain) and/or a light chain variable region (V_(L) domain) of theantibodies of the invention.

A person skilled in the art will appreciate that the proteins andpolypeptides of the invention, such as the light and heavy CDRs, thelight and heavy chain variable regions, antibodies, antibody fragments,and immunoconjugates, may be prepared in any of several ways well knownand described in the art, but are most preferably prepared usingrecombinant methods.

Nucleic acid fragments encoding the light and heavy chain variableregions of the antibodies of the invention can be derived or produced byany appropriate method, e.g., by cloning or synthesis.

Once nucleic acid fragments encoding the light and heavy chain variableregions of the antibodies of the invention have been obtained, thesefragments can be further manipulated by standard recombinant DNAtechniques, for example to convert the variable region fragments intofull length antibody molecules with appropriate constant region domains,or into particular formats of antibody fragment discussed elsewhereherein, e.g., Fab fragments, scFv fragments, etc. Typically, or as partof this further manipulation procedure, the nucleic acid fragmentsencoding the antibody molecules of the invention are generallyincorporated into an appropriate expression vector in order tofacilitate production of the antibodies of the invention.

Possible expression vectors include but are not limited to cosmids,plasmids, or modified viruses (e.g., replication defective retroviruses,adenoviruses and adeno-associated viruses), so long as the vector iscompatible with the host cell used. The expression vectors are “suitablefor transformation of a host cell”, which means that the expressionvectors contain a nucleic acid molecule of the invention and regulatorysequences selected on the basis of the host cells to be used forexpression, which are operatively linked to the nucleic acid molecule.Operatively linked is intended to mean that the nucleic acid is linkedto regulatory sequences in a manner that allows expression of thenucleic acid.

The invention therefore contemplates a recombinant expression vectorcontaining a nucleic acid molecule of the invention, or a fragmentthereof, and the necessary regulatory sequences for the transcriptionand translation of the protein sequence encoded by the nucleic acidmolecule of the invention.

Suitable regulatory sequences may be derived from a variety of sources,including bacterial, fungal, viral, mammalian, or insect genes and arewell known in the art. Selection of appropriate regulatory sequences isdependent on the host cell chosen as discussed below, and may be readilyaccomplished by one of ordinary skill in the art. Examples of suchregulatory sequences include: a transcriptional promoter and enhancer orRNA polymerase binding sequence, a ribosomal binding sequence, includinga translation initiation signal. Additionally, depending on the hostcell chosen and the vector employed, other sequences, such as an originof replication, additional DNA restriction sites, enhancers, andsequences conferring inducibility of transcription may be incorporatedinto the expression vector.

The recombinant expression vectors of the invention may also contain aselectable marker gene that facilitates the selection of host cellstransformed or transfected with a recombinant molecule of the invention.

The recombinant expression vectors may also contain genes that encode afusion moiety that provides increased expression of the recombinantprotein; increased solubility of the recombinant protein; and aid in thepurification of the target recombinant protein by acting as a ligand inaffinity purification (for example appropriate “tags” to enablepurification and/or identification may be present, e.g., His tags or myctags).

Recombinant expression vectors can be introduced into host cells toproduce a transformed host cell. The terms “transformed with”,“transfected with”, “transformation” and “transfection” are intended toencompass introduction of nucleic acid (e.g., a vector) into a cell byone of many possible techniques known in the art. Suitable methods fortransforming and transfecting host cells can be found in Sambrook etal., 1989 (Sambrook, Fritsch and Maniatis, 9i Molecular Cloning: ALaboratory Manual, 2nd Ed., Cold Spring Harbor Press, Cold SpringHarbor, N.Y., 1989) and other laboratory textbooks.

Suitable host cells include a wide variety of eukaryotic host cells andprokaryotic cells. For example, the proteins of the invention may beexpressed in yeast cells or mammalian cells. HEK 293E cells areparticularly preferred. In addition, the proteins of the invention maybe expressed in prokaryotic cells, such as Escherichia coli.

Given the teachings provided herein, promoters, terminators, and methodsfor introducing expression vectors of an appropriate type into plant,avian, and insect cells may also be readily accomplished.

Alternatively, the proteins of the invention may also be expressed innon-human transgenic animals such as, rats, rabbits, sheep and pigs.

The proteins of the invention may also be prepared by chemical synthesisusing techniques well known in the chemistry of proteins such as solidphase synthesis.

N-terminal or C-terminal fusion proteins comprising the antibodies andproteins of the invention conjugated to other molecules, such asproteins, may be prepared by fusing through recombinant techniques. Theresultant fusion proteins contain an antibody or protein of theinvention fused to the selected protein or marker protein, or tagprotein as described herein. The antibodies and proteins of theinvention may also be conjugated to other proteins by known techniques.For example, the proteins may be coupled using heterobifunctionalthiol-containing linkers as described in WO 90/10457,N-succinimidyl-3-(2-pyridyldithio-proprionate) or N-succinimidyl-5thioacetate.

A yet further aspect provides an expression construct or expressionvector comprising one or more of the nucleic acid fragments or segmentsor molecules of the invention. Preferably the expression constructs orvectors are recombinant. Preferably said constructs or vectors furthercomprise the necessary regulatory sequences for the transcription andtranslation of the protein sequence encoded by the nucleic acid moleculeof the invention.

A yet further aspect provides a host cell or virus comprising one ormore expression constructs or expression vectors of the invention. Alsoprovided are host cells or viruses comprising one or more of the nucleicacid molecules of the invention. A host cell or virus expressing anantibody of the invention forms a yet further aspect. Suitable hostcells include, but are not limited to HEK293E cells.

A yet further aspect of the invention provides a method of producing anantibody of the present invention comprising a step of culturing thehost cells of the invention. Preferred methods comprise the steps of (i)culturing a host cell comprising one or more of the recombinantexpression vectors or one or more of the nucleic acid sequences of theinvention under conditions suitable for the expression of the encodedantibody or protein; and optionally (ii) isolating or obtaining theantibody or protein from the host cell or from the growthmedium/supernatant. Such methods of production may also comprise a stepof purification of the antibody or protein product and/or formulatingthe antibody or product into a composition including at least oneadditional component, such as a pharmaceutically acceptable carrier orexcipient.

In embodiments when the antibody or protein of the invention is made upof more than one polypeptide chain (e.g., certain fragments such as Fabfragments), then all the polypeptides are preferably expressed in thehost cell, either from the same or a different expression vector, sothat the complete proteins, e.g., binding proteins of the invention, canassemble in the host cell and be isolated or purified therefrom.

In another aspect, the invention provides a method of binding IPPA-1a,comprising contacting a composition comprising HPA-1a with an antibodyof the invention, or an immunoconjugate thereof.

In yet another aspect, the invention provides a method of detectingHPA-1a, comprising contacting a composition suspected of containingHPA-1a with the antibody of the invention, or an immunoconjugatethereof, under conditions effective to allow the formation ofHPA-1a/antibody complexes and detecting the complexes so formed.

The antibodies of the invention may also be used to produce furtherantibodies that bind to HPA-1a. Such uses involve for example theaddition, deletion, substitution or insertion of one or more amino acidsin the amino acid sequence of a parent antibody to form a new antibody,wherein said parent antibody is one of the antibodies of the inventionas defined elsewhere herein, and testing the resulting new antibody toidentify antibodies that bind to HPA-1a. Such methods can be used toform multiple new antibodies that can all be tested for their ability tobind HPA-1a. Preferably said addition, deletion, substitution orinsertion of one or more amino acids takes place in one or more of theCDR domains.

Such modification or mutation to a parent antibody can be carried out inany appropriate manner using techniques well known and documented in theart, for example by carrying out methods of random or directedmutagenesis. If directed mutagenesis is to be used then one strategy toidentify appropriate residues for mutagenesis utilizes the resolution ofthe crystal structure of the binding protein-antigen complex, e.g,, theAb-Ag complex, to identify the key residues involved in the antigenbinding. Subsequently, those residues can be mutated to enhance theinteraction. Alternatively, one or more amino acid residues can simplybe targeted for directed mutagenesis and the effect on binding to HPA-1aassessed.

Random mutagenesis can be carried out in any appropriate way, e.g., byerror-prone PCR, chain shuffling or mutator E. coli strains.

Thus, one or more of the V_(H) domains of the invention can be combinedwith a single V_(L) domain or a repertoire of V_(L) domains from anyappropriate source and the resulting new antibodies tested to identifyantibodies specific for HPA-1a. Conversely, one or more of the V_(L)domains of the invention can be combined with a single V_(H) domain orrepertoire of V_(H) domains from any appropriate source and theresulting new antibodies tested to identify antibodies that bind toHPA-1a.

Similarly, one or more, or preferably all three CDRs of the V_(H) and/orV_(L) domains of the invention can be grafted into a single V_(H) and/orV_(L) domain or a repertoire of V_(H) and/or V_(L) domains, asappropriate, and the resulting new antibodies tested to identifyantibodies that bind to HPA-1a.

Methods of carrying out the above described manipulation of amino acidsand protein domains are well known to a person skilled in the art. Forexample, said manipulations could conveniently be carried out by geneticengineering at the nucleic acid level wherein nucleic acid moleculesencoding appropriate binding proteins and domains thereof are modifiedsuch that the amino acid sequence of the resulting expressed protein isin turn modified in the appropriate way.

The new antibodies produced by these methods will preferably haveimproved functional properties, e.g. a higher or enhanced affinity (orat least an equivalent affinity) for HPA-1a as the parent antibodies,and can be treated and used in the same way as the antibodies of theinvention as described elsewhere herein (e.g., for therapy, diagnosis,in compositions etc.), Alternatively, or additionally, the newantibodies will have one or more other improved functional properties asdescribed elsewhere herein.

New antibodies produced, obtained or obtainable by these methods form ayet further aspect of the invention.

Testing the ability of one or more antibodies to bind to HPA-1a can becarried out by any appropriate method, which are well known anddescribed in the art. Suitable methods are also described in theExamples section.

The invention also provides a hybridoma secreting the 26.4 antibody(e.g. the hybridoma DL18BL26.4H described in the Example section). Incertain embodiments, the invention provides an antibody secreted by sucha hybridoma (or an antibody which competes with such an antibody forbinding to HPA-1a ).

The invention also provides a range of conjugated antibodies andfragments thereof in which the anti-HPA-1a antibody is operativelyattached to at least one other therapeutic or diagnostic agent. The term“immunoconjugate” is broadly used to define the operative association ofthe antibody with another effective agent and is not intended to refersolely to any type of operative association, and is particularly notlimited to chemical “conjugation”. Recombinant fusion proteins areparticularly contemplated. So long as the delivery or targeting agent isable to bind to the target and the therapeutic or diagnostic agent issufficiently functional upon delivery, the mode of attachment will besuitable.

The invention also provides an antibody as defined herein coupled to asolid support (e.g. a microsphere).

Formulations (compositions) comprising one or more antibodies of theinvention in admixture with a suitable diluent, carrier or excipientconstitute a further aspect of the present invention. Such formulationsmay be for pharmaceutical use. Suitable diluents, excipients andcarriers are known to the skilled man.

The compositions according to the invention may be presented, forexample, in a form suitable for oral, nasal, parenteral, intravenal,topical or rectal administration.

The active compounds defined herein may be presented in the conventionalpharmacological forms of administration, such as tablets, coatedtablets, nasal sprays, solutions, emulsions, liposomes, powders,capsules or sustained release forms. Conventional pharmaceuticalexcipients as well as the usual methods of production may be employedfor the preparation of these forms.

Injection solutions may, for example, be produced in the conventionalmanner, such as by the addition of preservation agents, such asp-hydroxybenzoates, or stabilizers, such as EDTA. The solutions are thenfilled into injection vials or ampoules.

Nasal sprays may be formulated similarly in aqueous solution and packedinto spray containers, either with an aerosol propellant or providedwith means for manual compression.

The pharmaceutical compositions (formulations) of the present inventionare preferably administered parenterally. Parenteral administration maybe performed by subcutaneous, intramuscular or intravenous injection bymeans of a syringe, optionally a pen-like syringe. Alternatively,parenteral administration can be performed by means of an infusion pump.A further option is a composition which may be a powder or a liquid forthe administration of the peptide in the form of a nasal or pulmonalspray. As a still further option, the antibodies of the invention canalso be administered transdermally, e.g. from a patch, optionally aniontophoretic patch, or transmucosally, e.g. bucally.

Dosage units containing the antibodies preferably contain 0.1-10 mg, forexample 1-5 mg of the active agent. Other useful doses include, but arenot limited to, doses which achieve a plasma concentration of 0.1 to 1for example 0.5 IU/mL (0.08 μg/mL). Such a dose of 0.5 IU/mL (0.08μg/mL) may be achieved by intravenous administration of 2,000 IU.

The pharmaceutical compositions may additionally comprise further activeingredients as described above in the context of co-administrationregimens.

A further aspect of the present invention provides the anti-HPA-1aantibodies defined herein for use in therapy, in particular for use inthe treatment or prophylaxis of FNAIT. Thus, therapy includesprophylactic treatment.

HPA-1a negative (i.e. HPA-1bb) women may produce anti-HPA-1a antibodiesas a result of immunization with HPA-1a in connection with anon-compatible pregnancy (i.e. a pregnancy with a HPA-1a positivefetus). Such maternally produced anti-HPA-1a antibodies traverse theplacenta, bind fetal platelets and may accelerate platelet destruction,thereby causing FNAIT.

Without wishing to be bound by theory, it is believed that theantibodies of the present invention administered to such analloimmunized woman cross the placenta and compete with maternal HPA-1aantibodies for binding to the fetal platelets, thereby reducing plateletdestruction and thus treating FNAIT.

In the context of FNAIT treatment, preferably the antibody has a reducedor abolished effector function. For example, the Fc portion of animmunoglobulin (1 g) can be modified (or removed) in order toreduce/remove the effector function. Methods for doing so are known inthe art. Preferably, in the context of FNAIT treatment, the antibody hasan Fc portion which favours placental transfer and which has mutationswhich diminish (or abolish) its platelet destructive properties (e.g. asdiscussed in Mathiesen et al., Blood (2013) 122(7):1174-81).

In the context of FNAIT treatment, certain preferred subjects foradministration with an antibody of the invention are those known to becarrying a fetus that is already suffering from FNAIT, as determined by,for example a platelet count in the fetus.

The antibodies of the present invention may also be used to preventFNAIT, i.e. may be used in prophylactic treatments. In certainembodiments of such prophylactic treatments, the antibodies of theinvention may be administered to a woman who is already pregnant,preferably to a pregnant woman known to be HPA-1a negative, morepreferably a woman already pregnant with an incompatible pregnancy (i.e.the mother is HPA-1a negative and the fetus is HPA-1a positive).

It has been found that alloimmunization with HPA-1a can also occur inconnection with delivery of a non-compatible fetus (baby), HPA-1astimulation at delivery can be the first HPA-1a stimulus that the motherhas received (i.e. there may have been no alloimmunization duringpregnancy). Thus, in certain embodiments, antibodies of the inventionare administered to mother at delivery or shortly after delivery,preferably within 72 hours of delivery.

Without wishing to be bound by theory, anti-HPA-1a antibodies of thepresent invention administered to a mother in connection with delivery(or otherwise at risk of alloimmunization) would bind to HPA-1a onHPA-1a positive fetal (baby's) platelets entering the maternalcirculation and destroy the HPA-1a positive platelets thereby preventingstimulation of the mother's immune system by the fetus'/baby's HPA-1abearing platelets. Accordingly, alloimmunization is prevented, and FNAITdoes not occur. An analogous mechanism prevents alloimmunization inconnection with fetal trophoblasts or other trophoblast materialentering the maternal circulation.

As described above, in some embodiments the anti-HPA-1a antibodies ofthe present invention have the ability to inhibit the binding of theanti-HPA-1a antibody SZ21 to αVβ3 integrin. Without wishing to be boundby theory, the ability of an antibody of the invention to stably bind toαVβ3 integrin and to be able to inhibit the binding of other anti-HPA-1aantibodies to αVβ3 integrin indicates that such antibodies would haveutility in the prevention of alloimmunization in connection with fetaltrophoblasts or other trophoblast material entering the maternalcirculation.

In the case where alloimmunization in connection with delivery isprevented, a subsequent non-compatible pregnancy can be protected fromFNAIT.

Thus, in a further aspect, the invention also provides the anti-HPA-1aantibodies defined herein for use in preventing alloimmunization withHPA-1a in a subject.

Ghevaert et al. (Blood, 122: 313-320 (2013)) discusses the use of ananti-HPA-1a antibody in the treatment and prophylaxis of FNAIT.

In some embodiments, particularly in the context of FNAIT prophylaxis,anti-HPA-1a IgG antibodies are preferably glycosylated. In someembodiments, particularly in the context of FNAIT prophylaxis,anti-HPA-1a IgG antibodies are preferably not fucosylated (i.e.preferably not modified with a fucose group).

Antibodies of the present invention bind specifically to HPA-1a (i.e. donot cross-react with the alloantigen HPA-1b). Thus, the antibodies ofthe invention can be used to determine whether a subject (preferably afemale subject) is HPA-1a positive or HPA-1a negative.

Accordingly, in a further aspect, the invention provides a method foranalysing for the presence or absence of HPA-1a in a sample (preferablya sample containing platelets) that has been obtained from a subject,said method comprising the steps of

-   -   (a) contacting said sample with an antibody of the invention        which binds specifically to HPA-1a; and    -   (b) analysing for the presence or absence of anti-HPA-1a        antibody-HPA-1a (antigen) complexes.

The presence of anti-HPA-1a antibody-HPA-1a (antigen) complexesindicates the presence of HPA-1a in the sample. The absence ofanti-HPA-1a antibody-HPA-1a (antigen) complexes indicates the absence ofHPA-1a in the sample. Thus, the present invention provides a method forHPA-1 phenotyping. Suitable methods for analysing for (i.e. determining)for the presence of HPA-1a antibody-HPA-1a (antigen) complexes are knownin the art. In one embodiment, whole blood flow cytometry is used,preferably in such embodiments an antibody of the invention (e.g. anIgG₁ form thereof) is conjugated to a fluorescent dye. In someembodiments the whole blood is peripheral blood, preferably obtainedfrom subjects no more than 10 days before it is used. In someembodiments whole blood cytometry is used in accordance with theexperimental examples herein.

A method for analysing for the presence or absence of HPA-1a in a sampleas described above can be used to identify women who might benefit fromthe prophylactic treatments described herein.

Several prospective studies found that high levels of maternalanti-HPA-1a antibodies correlate with low platelet count in the newborn.Thus, quantitation of anti-HPA-1a antibodies can be used as predictivefactor of the degree of thrombocytopenia in the newborn. Currently usedreference material for anti-HPA-1a antibody quantitation was prepared bythe National Institute of Biological Standards and Control (NIBSC). ThisNIBSC standard contains plasma from six HPA-1a immunized donors and itssupply is dependent on the availability of such donors. Replacingpolyclonal sera with a recombinant antibody would provide a relativelycheap, standardized, highly specific and unlimited source of anti-HPA-1aantibody to be used as a control reference reagent.

Thus, in a further aspect, the present invention provides the use of anantibody of the present invention as a reference standard forquantifying anti-HPA-1a antibodies (maternally produced) in a sample(e.g. a whole blood or plasma sample). Preferably said referencestandard is used in a MAIPA (monoclonal antibody immobilization ofplatelet antigens) assay to quantify anti-HPA-1a antibodies in a sample.

Alternatively viewed the present invention provides a method of treatingor preventing FNAIT which method comprises administering to a patient inneed thereof a therapeutically or prophylactically effective amount ofan antibody of the invention as defined herein.

A therapeutically or prophylactically effective amount will bedetermined based on the clinical assessment and can be readilymonitored.

Further alternatively viewed, the present invention provides the use ofan antibody of the invention as defined herein in the manufacture of amedicament for treating or preventing FNAIT.

Subjects treated in accordance with the present invention willpreferably be humans, more preferably female humans (e.g. pregnantfemale subjects).

The compositions and methods and uses of the present invention may beused in combination with other therapeutics and diagnostics. In terms ofbiological agents, preferably diagnostic or therapeutic agents, for use“in combination” with an anti-HPA-1a antibody in accordance with thepresent invention, the term “in combination” is succinctly used to covera range of embodiments. The “in combination” terminology, unlessotherwise specifically stated or made clear from the scientificterminology, thus applies to various formats of combined compositions,pharmaceuticals, cocktails, kits, methods, and first and second medicaluses.

The “combined” embodiments of the invention thus include, for example,where the anti-HPA-1a of the invention is a naked antibody and is usedin combination with an agent or therapeutic agent that is notoperatively attached thereto. In other “combined” embodiments of theinvention, the anti-HPA-1a antibody of the invention is animmunoconjugate wherein the antibody is itself operatively associated orcombined with the agent or therapeutic agent. The operative attachmentincludes all forms of direct and indirect attachment as described hereinand known in the art.

The invention further includes kits comprising one or more of theantibodies, immunoconjugates or compositions of the invention or one ormore of the nucleic acid molecules encoding the antibodies of theinvention, or one or more recombinant expression vectors comprising thenucleic acid sequences of the invention, or one or more host cells orviruses comprising the recombinant expression vectors or nucleic acidsequences of the invention. Preferably said kits are for use in themethods and uses as described herein, e.g., the therapeutic, diagnosticor imaging methods as described herein, or are for use in the in vitroassays or methods as described herein. The antibody in such kits maypreferably be an antibody conjugate as described elsewhere herein, e.g.,may be conjugated to a detectable moiety or may be an irnmumoconjugate.Preferably said kits comprise instructions for use of the kitcomponents, for example in diagnosis. Preferably said kits are fordiagnosing or treating diseases as described elsewhere herein, andoptionally comprise instructions for use of the kit components todiagnose or treat such diseases.

The antibodies of the invention as defined herein may also be used asmolecular tools for in vitro or in vivo applications and assays. As theantibodies have an antigen binding site, these can function as membersof specific binding pairs and these molecules can be used in any assaywhere the particular binding pair member is required.

Thus, yet further aspects of the invention provide a reagent thatcomprises an antibody of the invention as defined herein and the use ofsuch antibodies as molecular tools, for example in in vitro or in vivoassays.

Table of Nucleotide and Amino Acid Sequences Disclosed Herein and TheirSequence Identifiers (SEQ ID NOs)

All nucleotide sequences are recited herein 5′ to 3′ in line withconvention in this technical field.

TABLE 1 SEQ ID NO: Description Sequence 26.4 antibody 1 VH domaincaggtacagttgcagcagtcaggtccaggac (nt) tggtgaagccctcgcagaccctgtcactcacctgtgccatctceggggacagtgtcagcagc aacagtgctgcttggaactggatcaggcagtccccatcgagaggccttgagtggctgggaag gacatacttcaggtccaactggtacaatgattatgcagcatctotgaaaagtegaataacca tcaaccaagacacatccaagaaccagctctccctgcagctgaactctgtgactcccgaggac acggctatgtattactgtgcaagagatggggcctggggtggcagcagctggtggccaggcct tcctcaccactactactctggtatgacgtctggggccaggggaccacggtcaccgtctcctc a 2 VL domaingaaattgtgttgacacagtctccagccaccc (nt) tgtcattgtctccaggggaaagagccaccotctcctgcagggccagtcagagtgttagcagc tacttagcctggtaccaacagaagcctggccaggctcccaggctcctcatctatgatgcatc caaaagggccactggcatcccagccaggttcagtggcagtgggtctgggacagacttcagtc tcaccatcagaagcctcgagcctgaagattttgcagtttattactgtcaacagcgtagcgac tggcagggactcactttcggcggagggaccaaggtggagatcaaa 3 VH domain QVQLQQSGPGLVKPSQTLSLICAISGDSVSS (aa)NSAAWNWIRQSPSRGLEWLGRTYFRSNWYND YAASVKSRITINQDTSKNQLSLQLNSVTPEDTAMYYCARDGAWGGSSWWPGLPHHYYSGMDV WGQGTTVTVSS 4 VL domainEIVLTQSPATLSLSPGERATLSCRASQSVSS (aa) YLAWYQQKPGQAPRLLIYDASKRATGIPARFSGSGSGTDFSLTIRSLEPEDFAVYYCQQRSD WQGLTFGGGTKVEIK 5 Heavy CDR1 GDSVSSNSAA6 Heavy CDR2 TYFRSNWYN 7 Heavy CDR3 ARDGAWGGSSWWPGLPHHYYSGMDV 8Light CDR1 QSVSSY 9 Light CDR2 DAS 10 Light CDR3 QQRSDWQGLT 11 Heavy FR1QQLQQSGPGLVKPSQTLSLTCAIS 12 Heavy FR2 WNWIRQSPSRGLEWLGR 13 Heavy FR3DYAASVKSRITINQDISKNQLSLQLNSVTPE DTAMYYC 14 Heavy FR4 WGQGTTVTVSS 15Light FR1 EIVLTQSPATLSLSPGERATLSCRAS 16 Light FR2 LAWYQQKPGQAPRLLIY 17Light FR3 KRATGIPARFSGSGSGTDFSLTIRSLEPEDF AVYYC 18 Light FR4 FGGGTKVEIK19 IgG1 heavy CAGGTGCAGCTGCAGCAGTCCGGCCCTGGGC chain (nt)TGGTGAAGCCTAGCCAGACCCTGTCCCTGAC ATGCGCCATCTCAGGCGACAGCGTGAGCTCCAACTCTGCCGCTTGGAATTGGATTAGACAGA GCCCATCCCGCGGGCTGGAGTGGCTGGGACGGACCTACTTCAGAAGCAACTGGTACAATGAC TATGCCGCTTCCGTGAAGTCTCGGATCACCATTAACCAGGATACATCTAAAAATCAGCTGAG TCTGCAGCTGAACTCAGTGACTCCCGAAGACACCGCCATGTACTATTGTGCTAGGGATGGCG CTTGGGGCGGGTCTAGTTGGTGGCCAGGACTGCCCCACCATTACTATAGCGGCATGGACGTG TGGGGACAGGGCACCACAGTGACAGTGTCAAGCGCCAGCACTAAGGGCCCTTCCGTGTTTCC TCTGGCTCCATCCTCTAAATCTACAAGTGGAGGCACTGCCGCTCTGGGGTGTCTGGTGAAGG ATTATTTCCCTGAGCCAGTGACTGTGAGTTGGAACTCAGGCGCCCTGACTAGCGGGGTGCAC ACCTTTCCCGCTGTGCTGCAGAGTTCAGGGCTGTACAGCCTGAGCTCCGTGGTGACCGTGCC TTCTAGTTCACTGGGAACTCAGACCTATATCTGCAACGTGAATCACAAGCCTTCTAATACAA AAGTGGACAAGAAAGTGGAGCCAAAGAGTTGTGATAAAACACATACTTGCCCTCCCTGCCCT GCCCCTGAACTGCTGGGCGGCCCAAGCGTGTTCCTGTTTCCACCCAAGCCCAAAGATACACT GATGATTAGCCGGACTCCGGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAGGATCCTG AAGTGAAGTTCAACTGGTACGTGGACGGCGTGGAAGTGCATAATGCCAAGACCAAACCACGG GAGGAACAGTACAACTCTACATATAGAGTGGTGAGTGTGCTGACTGTGCTGCACCAGGATTG GCTGAACGGGAAAGAGTATAAGTGCAAAGTGAGCAATAAGGCCCTGCCTGCTCCAATCGAGA AAACCATTTCCAAGGCCAAAGGACAGCCCAGGGAACCTCAGGTGTACACACTGCCCCCTAGT CGCGACGAGCTGACTAAGAACCAGGTGTCTCTGACCTGTCTGGTGAAAGGCTTCTATCCATC CGATATCGCTGTGGAGTGGGAATCTAATGGGCAGCCCGAAAACAATTACAAGACCACACCAC CCGTGCTGGACAGCGATGGATCCTTCTTTCTGTATTCAAAGCTGACTGTGGACAAAAGCCGG TGGCAGCAGGGCAACGTGTTTAGCTGTTCCGTGATGCATGAGGCTCTGCACAATCATTACAC CCAGAAGTCTCTGAGTCTGTCACCCGGGAAA TGA 20IgG1 GAGATCGTGCTGACTCAGTCTCCAGCCACCC light chainTGTCCCTGTCTCCAGGAGAACGGGCCACTCT (kappa) GTCTTGCAGAGCTAGTCAGTCAGTGAGCTCC(nt) TACCTGGCCTGGTATCAGCAGAAGCCAGGAC AGGCTCCCCGGCTGCTGATCTACGACGCCTCCAAAAGGGCTACAGGCATTCCCGCTCGCTTC AGCGGCTCCGGGTCTGGAACAGATTTTTCCCTGACTATCAGAAGCCTGGAGCCTGAAGACTT CGCCGTGTACTATTGCCAGCAGAGATCTGATTGGCAGGGGCTGACCTTTGGCGGGGGAACAA AGGTGGAGATCAAAAGGACCGTGGCCGCTCCAAGCGTGTTCATCTTTCCCCCTAGCGACGAA CAGCTGAAGTCAGGGACAGCCAGCGTGGTGTGCCTGCTGAACAATTTCTACCCCCGCGAGGC CAAGGTGCAGTGGAAAGTGGATAACGCTCTGCAGAGTGGAAATTCACAGGAGAGCGTGACTG AACAGGACTCCAAGGATTCTACCTATAGTCTGTCTAGTACCCTGACACTGAGCAAAGCCGAC TACGAGAAGCACAAAGTGTATGCTTGCGAAGTGACACATCAGGGCCTGTCAAGCCCTGTGAC TAAGAGCTTCAACCGGGGCGAGTGTTGA 21IG1 heavy QVQLQQSGPGLVKPSQTLSLTCATSGDSVSS chain (aa)NSAAWNWIRQSPSRGLEWLGRTYFRSNWYND YAASVKSRITINQDTSKNQLSLQLNSVTPEDTAMYYCARDGAWGGSSWWPGLPHHYYSGMDV WGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPTEKTTSKAKGQPREPQVYTLPPS RDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 22 IgG1EIVLTQSPATLSLSPGERATLSCRASQSVSS light chainYLAWYQQKPGQAPRLLIYDASKRATGIPARF (kappa) SGSGSGTDFSLTIRSLEPEDFAVYYCQQRSD(aa) WQGLTFGGGTKVEIKRTVAAPSVFIFPPSDE QLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSTDVTKSFNRGEC 23IgG3 heavy CAGGTGCAGCTGCAGCAGTCCGGGCCAGGAC chain (nt)TGGTGAAACCCTCACAGACACTGAGCCTGAC TTGTGCCATCAGTGGCGATTCAGTGAGCTCCAACAGCGCCGCTTGGAATTGGATTAGGCAGA GTCCTTCACGCGGACTGGAATGGCTGGGCCGGACCTACTTCAGATCCAACTGGTACAATGAC TATGCCGCCAGCGTGAAGTCCCGGATCACAATTAACCAGGATACTTCCAAAAATCAGCTGTC TCTGCAGCTGAACAGTGTGACCCCAGAGGACACAGCCATGTACTATTGCGCCAGAGATGGGG CTTGGGGCGGGTCTAGTTGGTGGCCAGGCCTGCCCCACCATTACTATAGCGGGATGGACGTG TGGGGACAGGGAACCACAGTGACCGTGAGCAGCGCCTCAACCAAAGGGCCTAGCGTGTTTCC TCTGGCTCCATGCAGCAGGTCCACTTCTGGAGGCACCGCCGCTCTGGGATGTCTGGTGAAGG ACTATTTCCCCGAACCTGTGACCGTGTCTTGGAACAGTGGGGCCCTGACCTCTGGAGTGCAC ACATTTCCCGCTGTGCTGCAGTCCTCTGGACTGTACAGCCTGAGTTCAGTGGTGACCGTGCC AAGCTCCTCTCTGGGCACACAGACTTATACCTGTAACGTGAATCACAAGCCCAGCAATACAA AGGTGGACAAACGGGTGGAGCTGAAAACACCTCTGGGCGATACTACCCATACTTGCCCACGG TGTCCAGAGCCCAAAAGCTGTGACACCCCTCCCCCATGCCCAAGATGTCCTGAACCAAAATC TTGTGATACACCCCCTCCATGCCCTAGGTGTCCCGAGCCTAAGAGTTGTGACACTCCCCCTC CATGTCCTAGATGTCCTGCTCCGGAACTGCTGGGCGGTCCGAGCGTGTTTCTGTTCCCGCCG AAACCGAAAGATACCCTGATGATTAGTCGCACGCCGGAAGTTACCTGCGTGGTTGTGGATGT GAGCCATGAAGACCCGGAAGTTCAGTTTAAATGGTATGTGGATGGTGTTGAAGTGCACAACG CAAAAACCAAACCGCGTGAAGAACAGTACAATAGCACGTTCCGCGTTGTGTCTGTTCTGACC GTGCTGCATCAGGATTGGCTGAACGGCAAAGAATACAAATGTAAAGTTTCTAACAAAGCACT GCCGGCGCCGATTGAAAAAACGATCAGTAAAACCAAGGGTCAGCCGCGTGAACCGCAGGTGT ACACCCTGCCGCCGAGCCGTGAAGAAATGACGAAAAACCAAGTTAGTCTGACCTGCCTGGTG AAAGGCTTTTACCCGAGCGATATTGCAGTGGAATGGGAAAGCTCTGGTCAGCCGGAAAACAA TTATAATACCACGCCGCCGATGCTGGATAGTGATGGCAGCTTTTTCCTGTATAGTAAACTGA CCGTTGATAAAAGCCGTTGGCAGCAGGGTAACATCTTTAGTTGCAGCGTGATGCATGAAGCG CTGCACAATCGCTTCACCCAGAAATCTCTGAGTCTGAGCCCGGGCAAAGGTAAATAA 24 IgG3 GAGATCGTGCTGACTCAGTCTCCAGCCACCClight chain TGTCCCTGTCTCCAGGAGAACGGGCCACTCT (kappa)GTCTTGCAGAGCTAGTCAGTCAGTGAGCTCC (nt) TACCTGGCCTGGTATCAGCAGAAGCCAGGACAGGCTCCCCGGCTGCTGATCTACGACGCCTC CAAAAGGGCTACAGGCATTCCCGCTCGCTTCAGCGGCTCCGGGTCTGGAACAGATTTTTCCC TGACTATCAGAAGCCTGGAGCCTGAAGACTTCGCCGTGTACTATTGCCAGCAGAGATCTGAT TGGCAGGGGCTGACCTTTGGCGGGGGAACAAAGGTGGAGATCAAAAGGACCGTGGCCGCTCC AAGCGTGTTCATCTTTCCCCCTAGCGACGAACAGCTGAAGTCAGGGACAGCCAGCGTGGTGT GCCTGCTGAACAATTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAAGTGGATAACGCTCTG CAGAGTGGAAATTCACAGGAGAGCGTGACTGAACAGGACTCCAAGGATTCTACCTATAGTCT GTCTAGTACCCTGACACTGAGCAAAGCCGACTACGAGAAGCACAAAGTGTATGCTTGCGAAG TGACACATCAGGGCCTGTCAAGCCCTGTGACTAAGAGCTTCAACCGGGGCGAGTGTTGA 25 IgG3 QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSheavy chain NSAAWNWTRQSPSRGLEWLGRTYFRSNWYND (aa)YAASVKSRITINQDTSKNQLSLQLNSVTPED TAMYYCARDGAWGGSSWWPGLPHHYYSGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYT CNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRC PEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFK WYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK TKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDTAVEWESSGQPENNYNTTPPMLDS DGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGKGK 26 IgG3 EIVLTQSPATLSLSPGERATLSCRASQSVSS light chainYLAWYQQKPGQAPRLLIYDASKRATGIPARF (kappa) SGSGSGTDFSLTIRSLEPEDFAVYYCQQRSD(aa) WQGLTFGGGTKVEIKRTVAAPSVFTFPPSDE QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGECThe IgG nucleic acid sequences set forth in the above Table areoptimised for expression in HEK cells.

The invention will now be further described in the followingnon-limiting Examples with reference to the following drawings:

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B. isolation of HPA-1a -specific B-lymphoblasts. FIG. 1Aillustrates cell positive for CD22 were isolated by MACS from PBMCs ofan HPA-1a alloimmunized woman and labelled with FITC-conjugatedanti-human IgM, IgA and IgD antibodies. The CD22⁻IgM⁻IgD⁻IgA⁻ population(gated, 5.6% of CD22⁻ B cells), the IgG⁺ memory B cells, was identifiedand isolated by FACS. FIG. 1B illustrates HPA-1a -positive plateletswere labelled with CFSE, incubated with B-lymphoblasts from theB-lymphoblast culture secreting anti-HPA-1a antibodies, andplatelet-bound B-lymphoblasts (gated, 2% of CD45⁺ B-Iymphoblasts) wereisolated individually by FACS into 96 well U-bottom micro plates.Results are representative of at least three independent experiments.

FIGS. 2A and 2B. Binding of mAb 26.4 to HPA-1antigens on intactplatelets. FIG. 2A shows a binding of 26.4 to HPA-1aa and HPA-1bbplatelets analysed by flow cytometry. Platelets were incubated with 26.4cell culture supernatant or medium as a negative control.FITC-conjugated anti-human IgG was used to detect platelet-bound IgG.The results are presented as an overlay of histograms: relative numberof cells plotted against the fluorescence intensity. FIG. 2B illustratesthe 26.4 was tested against HPA-1aa and HPA-1bb platelets in MAIPAassay. Normal serum was used as a negative control. Samples were run induplicates. Presented are average absorbance values after backgroundsubtraction. Results are representative of at least three independentexperiments. B-lymphoblast and hybridoma derived 26.4, and recombinant26,4 IgG1and IgG3 performed alike.

FIG. 3. Nucleotide and amino acid sequence of mAb 26.4. Heavy and Lightchain V-regions compared with the most homologous germline sequences.Analyzed by IMGT/V-QUEST.

FIGS. 4A and 4B. SPR analysis of mAb binding to HPA-1antigens.Sensograms generated by binding of 26.4 IgG1as shown in FIG. 4A andSZ21as shown in FIG. 4B to the allbβ3 bearing the HPA-1a (black line) orHPA1b (dashed line) antigens immobilized to the sensor chip surface.Antibodies were used at a concentration of 20 μg/ml.

FIGS. 5A-5F. SPR analysis of mAb binding to HPA-1a on allbβ3 and αVβ3.Sensograms generated by binding of 26.4 IgG1 (black line) and B2G1(dashed) to HPA-1a on allbβ3 as shown in FIG. 5A and αVβ3 as shown inFIG. 5 Bimmobilized to the sensor chip surface. MAb samples were used inthree different concentrations (20 μg/ml, 10 μg/ml and 5 μg/ml); thehighest concentration is shown. Results are representative of the twoindependent experiments. FIG. 5C shows a relative binding response of26.4 and B2G1 to HPA-1a on allbβ3 and αVβ3. Binding response (RU) at theend of association period was calculated relative to 26.4 (26.4 RU weretaken as 100% for each integrin). Data presented are average RUgenerated by injection of three different concentrations of mAbs (20μg/ml, 10 μg/ml and 5 g/ml). FIG. 5D shows a percentage of 26.4 and B2G1bound to HPA-1a on allbβ3 and αVβ3 at the end of the dissociationperiod. The percentage of antibody bound at the end of the dissociationphase was calculated by dividing the RU at the end of dissociationperiod by the RU at the end of association period multiplied by 100%.Data presented are average percentage calculated from three differentconcentrations for each mAb.

To compare the capacity of 26.4 and B2G1 to inhibit binding of mAb SZ21to HPA-1a antigen, beads coupled with β3 integrin were preincubated withvarious concentrations of 26.4 or B2G1and subsequent binding ofFITC-conjugated SZ21 to HPA-1a antigen was evaluated by flow cytometry(FIGS. E and F). Relative fluorescence intensity=mean fluorescenceintensity of each sample (mean±SEM)—mean fluorescence intensity of beadscoupled with β3 integrin from HPA-1bb platelet lysate. Every sample wasrun in duplicate. The presented graphs represent four independentexperiments using beads coupled with β3 integrin from platelet lysate asshown in FIG. 5E or from trophoblast cell lysate as shown in FIG. 5F.

FIG. 6. Effect of mAb 26.4 on platelet aggregation. Blood samples fromHPA-1-genotyped donors (n=3 of each HPA-1 genotype) were preincubatedwith various concentrations of 26.4 IgG1 prior to addition of plateletactivator, Aggregation data for blood samples preincubated with 26.4 arepresented as percentage of platelet aggregation control.

FIGS. 7A and 7B. Monocyte phagocytosis of platelets opsonized with 26.4.Platelets from donors with known HPA-1 genotype (n=3 of each HPA-1genotype) were CMFDA labeled, sensitized with various concentrations of26.4 IgG1 or IgG3, and incubated with autologous monocytes. Afterremoval of adhered platelets, monocytes were stained with PE-conjugatedanti-CD14 antibody and analysed by flow cytometry. The CD14-positivepopulation was gated and the percentage of FITC-positive monocytes wasdefined as phagocytic activity (%). Data presented are averagephagocytic activity of monocytes from HPA-1a-homozygous donors (FIG. 7A)and from HPA-1ab donors (FIG. 7B).

FIGS. 8A and 8B. Illustration of a typical histogram for HPA-1phenotyping by whole blood flow cytometry using 26.4 conjugated to afluorescent dye. The population of platelets is gated in a dot plot(upper panel). Overlay of histograms show typical results forHPA-1a-positive (filled) and HPA-1a-negative platelets (lower panel).

FIG. 9. MAb26.4 preparation has a linearity and range comparable withthe commercially available polyclonal anti-HPA-1a NIBSC standard. Plotsgenerated by mean absorbance values for replicate doubling dilutions ofNIBSC and proposed mAb 26.4 IgG1 standards in MAIPA assay. Linearportions of the plots were used to determine the anti-HPA-1a activity ofthe samples.

FIG. 10. Anti-HPA-1a activities of samples A, B, C and D ininternational units per ml (IU/ml). The mean anti-HPA-1a activity valuefor each sample and standard deviation (CD) from three MAIPA assays werecalculated when NIBSC or mAb were used as standards.

FIG. 11. MAb 26.4 can inhibit binding of polyclonal anti-HPA-1a IgG toHPA-1a homozygous platelets. HPA-1aa platelets were reacted with variousconcentrations of 26.4 F(ab )₂fragment before adding polyclonalanti-HPA-1a IgG samples. Binding of anti-HPA-1a IgG to platelets wasmeasured by MAIPA. Uninhibited binding of polyclonal antibodies wastaken as maximum or 100% binding. Binding in the presence of 26.4F(ab′)₂ fragment is presented as a percentage of maximum binding. Dotsconnected by black lines represent binding of donor samples.

FIG. 12. Reactivity of murine mAbs specific to β₃ integrin withrecombinant β3 domain-deletion peptides analyzed by ELISA.Representative of two independent experiments. Experimental detailsprovided in Example 4.

FIG. 13. Reactivity of human mAbs specific to HPA-1a with recombinant β₃domain-deletion peptides analyzed by ELISA. Representative of twoindependent experiments, Experimental details provided in Example 4.

EXAMPLES Example 1

Generation and in Vitro Characterization of a Novel HumanHPA-1a-Specific Monoclonal Antibody

In this study, the aim was to develop a human mAb highly specific forthe HPA-1a that would be suitable for prophylactic, therapeutic andscreening purposes. An essential quality of such an antibody would behigh binding affinity to the HPA-1a and minimal reactivity with theHPA-1b counterpart. As described below, a fully human mAb was developedby immortalization of antigen specific memory B cells from anHPA-1a-negative woman who had developed anti-HPA-1a antibodies uponimmunization in connection with a non-compatible pregnancy (i.e. whereinthe fetus was HPA-1a positive).

Materials and Methods

Donor Material

Peripheral blood was donated by a woman who was HPA-1a immunized inconnection with pregnancy. She gave birth to two HPA-1a-positive babieswith severe thrombocytopenia and subcutaneous haemorrhages. The donatedblood sample was taken 4 weeks after delivery of the second child. Theplasma anti-HPA-1a antibody level was 150 IU/ml as measured byquantitative monoclonal antibody immobilization of platelet antigens(MAIPA) assay (Kiefel V, Santoso S, Weisheit M, Mueller-Eckhardt C.,Blood. 1987;70(6)1 722-6.).

Isolation of Memory B Lymphocytes

Peripheral blood mononuclear cells (PBMCs) were isolated bydensity-gradient centrifugation using Lymphoprep (Axis-Shield, Dundee,Scotland) according to the manufacturer's instructions. Memory B cellswere isolated based on the method of Traggiai et al. (Nat Med.2004;10(8):871-5.). Briefly, antibody labelled CD22⁺ cells were isolatedusing magnetic-activated cell sorting (MACS, Miltenyi Biotech, Germany),incubated with FITC-conjugated goat anti-human IgD, IgM and IgAantibodies (Dako, Denmark). The CD22⁺IgD⁻IgM⁻IgA⁻ cell population, IgG⁺memory B cells, was identified and isolated by fluorescent-activatedcell sorting (FACSAria BD Biosciences). Flow cytometry data was analysedby FlowJo software (TreeStar, Ashland, Oreg., USA).

EBV Transformation of Memory B Cells

Isolated memory B cells were seeded at 400 cells per well in 96 U-bottomcell culture plates and cultured in complete medium (lscove modifiedDulbecco medium (IMDM), 10% FBS and 100 Ul/ml Penicillin, 100 Ul/mlStreptomycin) with EBV-containing supernatant from a marmosetlymphoblast cell line B95.8 (ATCC number: VR-1492) and 0.6 μg/mlphosphorothioated CpG ODN2006 (15) (Integrated DNA technologies,Belgium) in humidified atmosphere at 37° C., 7.5% CO₂. After 2 weeks,the culture supernatants were tested for the presence of HPA-1a-specificIgG.

Selection of HPA-1a -Specific B-Lymphoblasts

HPA-1a-positive platelets were prepared from platelet rich plasma (PRP)(by pelleting) and labelled with carboxyfluorescein diacetatesuccinimidyl ester (CFSE; Invitrogen, Carlsbad, Calif.). Cells fromB-lymphoblast cultures secreting anti-HPA-1a IgG were stained withPerCP-conjugated anti-CD45 antibody (Caltag) and incubated withCFSE-labelled platelets. B-lymphoblasts binding HPA-1a-positiveplatelets were sorted one cell per well into 96 well U-bottom cultureplates by FACS and cloned in the presence of gamma irradiated allogeneicPBMC (10,000 cells per well).

Generation and Detection of anti-HPA-1a IgG Secreting Hhybridomas

Clonal B-lymphoblasts were fused to a non-secreting mouse-humanheteromyeloma cell line K6H6/B5 (ATCC number: CRL-1823) at a 1:10 ratiousing stirring method with polyethylene glycol (P7306, Sigma-Aldrich).Fused cells were seeded into the wells of a 48-well plate and culturedin complete medium. Hypoxanthine, aminopterin and thymidine (HAT;Sigma-Aldrich) selection was initiated 24 hours after cell fusion andcontinued for 7 days. Hybridoma supernatants were screened foranti-HPA-1a IgG by MAIPA or flow cytometry, For the MAIPA, we used 50 μlof culture supernatant and mouse monoclonal anti-CD61 antibody cloneY2/51 (Dako, Denmark) as capture antibody. For the flow cytometry assay,2×10⁶ HPA-1a-positive platelets were incubated with 50 μl of cellculture supernatant, washed and stained with FITC-conjugated anti-humanIgG antibodies (Dako, Denmark). Positive cultures were further subcloned3 times to isolate stable anti-HPA-1a antibody-secreting hybridomas. TheIgG subclass of the mAb was tested by ELISA. Goat anti-human antibodies(Caltag) were used to coat the ELISA plate (Maxisorp, Nunc) andbiotin-conjugated mouse anti-human anti-IgG1, IgG2, IgG3 and lgG4 mAbswere used as detection antibodies (clones HP6069, HP6002, HP6047 andHP6025, respectively, Invitrogen).

MAIPA Assay

The MAIPA technique described in detail in Killie et al, 2010 wasfollowed (Killie et al. 2010. Quantitative MAIPA: Comparison ofdifferent MAIPA protocols. Transfusion and Apheresis Science 43:149-54). Briefly, washed platelets were incubated with human serum orhuman mAb followed by a mouse monoclonal anti-GPIIb-IIIa antibody, cloneY2/51 (Dako). Platelets were then lysed and supernatant was added to amicroplate precoated with anti-mouse IgG. Human antibodies bound toGPIIb-IIIa were detected with labelled anti-human IgG and a suitablesubstrate. National Institute of Biological Standards and Control(NIBSC) polyclonal anti-HPA-1a standard (Allen D etal. 2005.Collaborative study to establish the first international standard forquantitation of anti-HPA-1a. Vox Sanguinis 89:100-4) were used to createa linear standard curve for quantitative MAIPA. Levels of specificantibodies in the samples were calculated using a linear regressionequation.

Purification of IgG from Cell Culture Supernatant

The lgG fraction of cell culture supernatant was isolated by 40%saturated ammonium sulphate precipitation followed by Protein G affinitychromatography (Protein G Sepharose 4 FastFlow, GE Healthcare). Theeluted IgG was dialyzed against phosphate buffered saline (PBS) andconcentrated using Microcon centrifugal filter devices (Ultracel YM-50,Millipore),

Amplification and Sequencing of Ig Variable Region Genes

Total RNA was isolated from clonal B-lymphoblasts using the RNeasy MiniSpin kit (QIAgen, Hilden, Germany). cDNA was synthesised via reversetranscription using primers specific for the human igG constant regions.The resulting cDNA was used as a template for polymerase chain reaction(PCR) to amplify IgG variable heavy and light region genes (VH, Vλ andVκ). The genes were amplified in separate PCR reactions for theindividual heavy and light chain gene families, using sense primerspecific to one of the leader regions, and anti-sense primer to theheavy and light chains constant regions. The PCR products wereidentified using 1.5% agarose gel electrophoresis and cloned intopCR2.1-TOPO vectors (TOPO TA cloning kit, Invitrogen) followed bysequencing of plasmid minipreps (Miniprep kit, QIAGEN). Sequencingreactions were precipitated and run on a 3130xl Genetic Analyzer(Applied Biosystems) at the sequencing core facility at the Faculty ofHealth Sciences, UiT, The Arctic University of Norway.

Analysis of Ig Variable Region Genes and Mutations

The nucleotide sequences were analyzed in the internationalImMunoGeneTics (lMGT) database of human germline genes usingIMGT/V-QUESTprogram available at httb://www.imgt.org (Brochet X, Lefranc M-P,Giudicelli V. Nucleic Acids Research. 2008;36 (suppl 2):W503-W8).Affinity maturation process (antigen selective pressure) leads toclustering of replacement (R) mutations as opposed to silent (S)mutations within complementarity determining regions (CDRs), which bindthe antigen. The framework regions (FRs) maintain the antibody structureand accumulate S as opposed to R mutations. The multinomial distributionmodel was used to determine whether relative abundance of R mutation inCDRs and S mutations in FRs accumulated at a rate higher than predictedto occur by chance based on codon composition of the parent germlinesequence. Mutations were identified for framework regions (FRs) 1, 2 and3 and complementary determining regions (CDRs) 1and 2 and imported alongwith Ig corresponding germline sequences into JAVA applet athttp://www-stat.stanford.edu/immunodlobulin/for multinomial analysis(Lassos I S, et al., J Immunol. 2000;165(9):5122-6).

Generation of Recombinant anti-HPA-1a I G1and IgG3

Synthesis of the 26.4 Heavy and Light Chain Genes

The heavy and light chain variable region genes coding for antibody 26.4were synthesised by GenScript (Piscataway, N.J., USA) optimizing thecodon usage in the synthesised genes for high level antibody expressionin human cells. Two variants of the 26.4 heavy chain gene weresynthesised utilizing the γ1 and γ3 heavy chain constant regions.Restriction enzyme recognition sites Esp3I and EcoRI were inserted intothe flanks of the synthesised genes, for subsequent use in the cloningof the genes into the pFRIDA vector (modified pLNO vector—Norderhaug Let al., J Immunol Methods 204: 77-87).

Cloning of the Genes

Each of the 26.4 genes was supplied in the pUC57 vector. The pUC57vector containing the synthesised gene was digested with restrictionenzymes Esp3I and EcoRI (Fermentas, Burlington, Canada) and the DNAfragment corresponding to the size of the heavy or light chain wasisolated by agarose gel electrophoresis using the Qiagen Gelelute kit(Qiagen, Germany). The pFRIDA cloning vector was processed in the sameway by digestion with restriction enzymes Esp3I nd EcoRI, and subsequentisolation of the digested vector by agarose gel electrophoresis. Thedigested genes were ligated into the linearized vector using T4 DNAligase (NEB, USA) and then transformed into XL-10 GOLD competent cells(Stratagene, USA). Transformed cells where selected on ampicillincontaining growth agar. Bacterial colonies were selected by growing 14hours in ampicillin containing liquid media and vector DNA was isolatedusing plasmid minipreps. The vector DNA was verified to contain thecorrect insert by restriction enzyme analysis.

Transient Transfection of HEK293E Cells for Expression of Antibody 26.4

Five million HEK293E cells were added to 25 ml DMEM medium (BE12-614F,Lonza) supplemented with 10% FBS and 4 mM L-glutamine. Thecell-containing medium was transferred to a standard medium cell cultureflask (T75) and incubated for 18 hours in humidified atmosphere at 37°C., 5% CO₂. A transfection mixture was prepared by adding 5 μg vectorDNA (0.1 μg/ml) expressing the 26.4 light chain, 5 μg vector DNA (0.1μg/ml) expressing the desired 26.4 heavy chain (γ1 or γ3) and 375 μlRPMI into a test tube. The mixture was preheated to 80° C. and cooled to4° C. Polyethylenimine Max (PEI Max, 2 mg/ml; 24765-2, Polysciences Inc)was heated simultaneously, but cooled to RT in order to preventprecipitation. Of the PEI solution, 65 μl was added to the transfectionmixture before the tube was left to incubate at RT for 8 min. DMEMmedium (10% FBS, 4 mM L-glutamin) (3375 μl) was then added to the testtube. The medium from the cell culture flask with HEK293E adherent cellswas removed and replaced with the reaction mixture. The reaction mixturewas allowed to cover cells for 2 hours before adding 25 ml DMEM mediumsupplemented with 10% FBS and 4 mM L-glutamine. The transfected cellswere allowed to grow for 2-5 days before the supernatant was harvestedand tested for production of IgG. The concentration of human IgG₁ andIgG₃ in samples was quantitated by ELISA, with goat anti-human IgG Fc(Sigma) as coating and ALP-conjugated goat anti-human IgG Fc (Sigma) asdetection antibodies. Human IgG1and IgG3 (I 5154 and I 5654respectively, Sigma) were used as internal standards.

Surface Plasmon Resonance (SPR) Analysis

SPR technology was used to assess the binding properties of the mAbs(Biacore T200 instrument, Biacore AB, Uppsala, Sweden). The αllbβ3integrin was isolated from HPA-1aa and −1bb platelets by affinitychromatography as described previously (Bakchoul T, Meyer O, Agaylan A,Bombard S, Bein G, Sachs U J H, et al. Transfusion. 2007;47(8):1363-8.),using a sepharose (CNBr-activated Sepharose 4 Fast Flow, GE Healthcare)column coupled with mouse anti-β3 mAb (clone AP3, ATCC number HB-242).The integrin αVβ3 was obtained from Millipore (cat. No: CC1018). Theintegrin αVβ3 was purified from human placenta by affinitychromatography using immobilized monoclonal antibodies to αVβ3 integrin.A tissue detergent extract applied to the column was prepared aspreviously described (Belkin V M, Belkin A M, Koteliansky V E., TheJournal of Cell Biology, 1990; 111(5)2159-70). The purified βllbβ3integrins (HPA-1a and HPA-1b antigen carrying versions) and αVβ3 wereimmobilized to the surface of a CM5 sensor chip on three different flowcells (FCs) at a density of 400, 340 and 480 response units (RU)respectively using standard amine coupling chemistry. An FC treated withthe same chemicals but without protein was used as a reference surface.Purified monoclonal IgG samples (various concentrations) were injectedover the chip surface at a flow rate of 30 μl/min. An association stepof 120 sec was followed by a dissociation step of 120 sec. Regenerationof the sensor chip surface was accomplished using 10 mM Glycine-HCl (pH1.5). The experiments were performed at 25° C. The collected data wereanalysed using BiaEvaluation 2.0.1 software. The amount of theimmobilized β3 integrin available for antibody binding was measured byinjection of the anti-β3 mAb (clone SZ21) at a concentration of 20μg/ml. Around 80 RU on the αllbβ3-immobilized chip (FIG. 4B) and 25 RUon the αVβ3-immobilized chip (data not shown) have been generated. Allchemicals for the Biacore experiment were purchased from GE Healthcare.

Flow Cytometric Antibody Binding-Inhibition Assay

The capacity of mAb 26.4 to inhibit binding of mAb SZ21 to the HPA-1aepitope was evaluated using beads indirectly coupled with β3 integrinand compared to a previously described recombinant HPA-1a antibody B2G1(Garner, et al., (2000), British Journal of Haematology 108: 440-7;Griffin H, et al., (1995), Blood 86: 4430-6). First, Dynabeads M-270Epoxy (Life Technologies) were coupled with an anti-β3 antibody (cloneEPR2417Y, specific for C-terminal part of β3-integrin, Abcam, Cambridge,England) according to the manufacturer's instructions. Next, beads wereincubated with cell lysate from a trophoblast cell line expressingβ3-integrin (TCL-1 (Lewis M P, etas, (1996), Placenta 17: 137-46);genotyped HPA-1aa) or platelet lysate from HPA-1a positive plateletsover night at 4° C., to bind β3 integrin from cell lysates. Beads werewashed with RIPA buffer (Sigma) and incubated with various amounts (12.5ng, 25 ng, 50 ng, 100 ng and 200 ng) of 26.4 and B2G1 in RIPA buffer for15 min at RT. These amounts of antibody were incubated with beads in atotal volume of 200 μl. The concentrations were therefore 62,5, 125,250, 500 and 1000 ng/ml, respectively. After a washing step, beads wereincubated with 5 μl of FITC-conjugated mAb SZ21 (Beckman Coulter) in 200μl bead suspension for 15 min at RT in dark. After a washing step, beadswere resuspended in PBS, and analyzed by flow cytometry.

Platelet Aggregometry (Multiplate)

Impedance platelet aggregometry was used to assess the effect of mAbs onplatelet aggregation (Multiplate analyser, Roche, Basel, Switzerland).Study participants (n=3 of each HPA-1 genotype) were healthy volunteerswith known HPA-1 genotype who did not take any medications affectingplatelet function 10 days prior to blood collection. Whole blood sampleswere drawn by peripheral venipuncture into 3 ml tubes, containingrecombinant hirudin as anticoagulant. The blood was kept at RT and themeasurements were performed within 2 h from blood collection. The 480 μlblood samples were incubated with various mAb concentrations (20 μlvolume) for 5 min before the addition of platelet activator, thrombinreceptor activating peptide-6 (TRAP-6). Blood samples with addition of20 μl of PBS buffer were used to determine the individual plateletfunction triggered by TRAP-6. To test the effect of the 26.4 on plateletaggregation without platelet activator, the 0.9% sodium chloridesolution was used instead of the TRAP-6. Aggregation was continuouslyrecorded over 6 min in two independent measuring units per test.Increase of impedance due to the attachment of platelets to theelectrodes was detected and converted into arbitrary aggregation units(AU) plotted against the time. The aggregation was quantified by thearea under the curve (AUC) in aggregation units (AU×min). Platelet countin blood samples was measured using Sysmex XN-1000 Hematology analyzer.

Anti-HPA-1a -Mediated Platelet Phagocytosis by Monocytes Assay

Buffy coat was diluted 1:4 in phosphate-buffered saline (PBS) andlayered over Lymphoprep medium (Axis-Shield, Dundee, United Kingdom)followed by 15 min centrifugation at 700g without brakes. The interfacewas collected, and 40 mL 0.2% PBSA (0.2% bovine serum albumin in PBS)was added. PBMCs were pelleted by centrifugation at 300 g for 6 minutes.The platelets were pelleted from the supernatant by centrifugation at2000 g for 6 minutes and resuspended in 0.2% PBSA 0.3% EDTA. Monocyteswere isolated from PBMCs using RosetteSep Human Monocyte EnrichmentCocktail (StemCell Technologies, Vancouver, Canada) as describedpreviously (Ahlen M T, Husebekk A, Killie M K, Skogen B, Stuge T B.Blood. 2009;113(16):3838-44.) and adjusted to 2×10⁶cell/ml in 10%FBS-IMDM (BE12-722F, Lonza).

In 1 ml volume 10⁸ platelets were labelled with CellTracker Green CMFDA(5-chloromethyl fluorescein diacetate, C7025, Invitrogen) at 0.25 μMfinal concentration according to the manufacturer's instructions.CMFDA-stained platelets were adjusted to 2×10⁸/ml in 0.2% PBSA 0.3% EDTAand 50 μl were incubated with different concentrations of humanmonoclonal anti-HPA-1a IgG for 20 min at RT. After a washing step, 50 μlof monocytes were added resulting to a total volume of 100 μl andplatelet to monocyte ratio 100:1 in duplicate tubes and incubated at 37°C., in a 7.5% CO₂ humidified atmosphere for 2 h. The monocytes werepelleted by centrifugation at 300 g and incubated with 0.25%trypsin/EDTA solution (T4049, Invitrogen) for 2 min at 37° C. to removeextracellular adherent platelets. After a washing step, the cells werestained with PE-conjugated anti-CD14 antibody (Invitrogen) and analysedby flow cytometry. Human IgG1and IgG3 of irrelevant specificities wereused as assay negative controls.

Statistics

Sigma Plot 12.5 software (San Jose, Calif.) was used to presentaggregation and phagocytosis experimental data. GraphPad Prism 5software (San Diego, Calif.) was used to present flow cytometricantibody binding inhibition assay data.

Ethics

The study was approved by Regional Committee for Medical ResearchEthics, North-Norway, (approval no: 2009/1585 and 2013/126/REK). Allvolunteers who donated blood samples have signed a written informedconsent (Blood Bank, University Hospital of North Norway).

Results

Monoclonal IgG specific for HPA-1a was Generated by ImmortalisingHPA-1-Specific Memory B Cells

It was reasoned that B cells producing anti-HPA-1a-specific IgG may bepresent at elevated numbers in the circulation of women who have givenbirth to a child affected by FNAIT, and that an antibody derived from asingle HPA-1a -specific B cell may give rise to a limitless supply ofmonoclonal antibodies with this specificity. In order to isolateHPA-1a-specific IgG⁺ B cells, PBMCs were first isolated from an HPA-1aalloimmunized woman. Blood was drawn 4 weeks after delivery of an FNAITaffected child. To enrich for B cells we reacted about 40 million PBMCswith a monoclonal antibody specific for the pan B cell marker CD22 andpurified the sensitized cells from the PBMCs by magnetic-activated cellsorting (MACS). About 3 million CD22⁺B cells were recovered. To enrichfor IgG⁺ B cells, the CD22⁺ cells were reacted with fluorescentlylabeled polyclonal antibodies to human IgM, IgA and IgD (IgMAD) isotypesand the IgMAD⁻ cells were isolated by fluorescence-activated cellsorting (FACS). The IgMAD⁻ cells amounted to 5.6% of the CD22⁺ cells.(FIG. 1A). In a separate experiment the IgMAD⁺ population of CD22⁺ cellswas shown to consist of mostly IgG⁺ cells (data not shown). About 10⁵cells were isolated by FACS. To isolate HPA-1a-specific B cells from theFACS-isolated cells, our strategy was to immortalize the sorted cells bytransformation with the Epstein-Barr virus (EBV) and to screen fortransformed cells producing anti-HPA-1a antibodies. Therefore, thesorted cells were treated with culture supernatant containing EBV in thepresence of a polyclonal activator of memory B cells, CpGoligonucleotide (CpG 2006) to enhance transformation and divided in 240wells (about 400 cells per well) on rnicrotitre plates. After 2 weeks,27 B-lymphoblast cultures containing HPA-1a-specific antibodies wereidentified by MAIPA. After 7 additional days in culture, only half ofthe B-lymphoblast cultures retained production of specific antibodies.Cells from the culture secreting the highest amount of anti-HPA-1a lgGwere incubated with CFSE-stained HPA-1a-positive platelets. TheCFSE-positive lymphoblasts, 120 cells, were isolated individually byFACS (FIG. 1B) and expanded in culture. Notably, we observed muchnonspecific binding of platelets to HPA-1a-negative B-lymphoblasts, usedas a negative control; the negative control had almost the samefrequency of CFSE-positive lymphoblasts (data not shown). After 3 weeksof expansion, one clonal B-lymphoblast culture secreting HPA-1a-specificantibodies was identified and clone D18BL26.4 (also referred to hereinas 26,4 or mAb26.4) was established. The 26.4 antibody boundspecifically to HPA-1a -positive platelets (FIG. 2A and FIG. 2B). Ahybridoma cell line, D18BL26.4H, secreting anti-HPA-1a IgG was generatedby fusing cells from the 26.4 B-lymphoblasts to heteromyeloma cells (asdescribed in the method section). The secreted lgG subclass was found tobe IgG3 by ELISA.

Amplification of Ig Variable Region Gene and Sequence Analysis

To test for clonality of the D18BL26.4 cell line and to amplify the Igvariable gene sequences, first we isolated mRNA and synthesized cDNA byreverse transcription with primers specific for the human IgG constantregions. The resulting cDNA was used as a template to amplify lgGvariable heavy and light region genes (VH, VAλ and Vκ) in separate PCRreactions for each gene family. The two amplified PCR products ofapproximately 400 bp in size corresponded to VH6 and VK3 gene families,confirming the clonality of the cells (data not shown). The PCR productswere sequenced and the analysis of Ig variable gene sequences enabledidentification of the closest known germline genes and the V, D, and Jgene segments used during somatic recombination (FIG. 3). For the heavychain IGHV6-1*01, IGHD6-13*01and IGHJ6*02 gene segments were used andIGKV3-11*01 and IGKJ4*01 for the light chain.

Recombinant mAb 26.4 is Specific for and Binds Strongly to the HPA-1aAntigen

To facilitate exploration of mAb 26.4 function with different Igisotypes the gene encoding the Ig heavy-chain variable region inD18BL26.4 cells was combined with IgG1 (26.4G1) and IgG3 (26.4G3)constant domains in different expression constructs. The light-chainvariable region gene was combined with a kappa 1 constant domain in athird construct. One heavy-chain and the light-chain constructs wereexpressed in HEK293E cells following transient transfection. Typically,transfected cultures produced 26.4G₁and 26.4G₃ to the supernatants atconcentrations of 20-50 μg/ml and 5-20 μg/ml, respectively, as measuredby ELISA. Identical to the native 26.4, mAbs 26.4G1and 26.4G3 boundspecifically to HPA-1a-positive intact platelets when tested in flowcytometry and MAIPA (FIGS. 2A and 2B). No binding to HPA-1a-negativeplatelets was observed. All the experiments from this point were donewith recombinant 26.4, and the 26.4G1 version was used unless otherwisenoted.

The 26.4 bound specifically to HPA-1a -positive intact platelets whentested in flax cytometry (FC) and IVIAIPA. No binding to theHPA-1a-negative platelets was observed.

In order for more sensitive assessment of specificity, 26.4 binding topurified platelet integrin αllbβ3 was measured by surface plasmonresonance (SPR). In the surface plasmon resonance (SPR) system, the 26.4bound exclusively to αllbβ3 from HPA-1aa individuals; there was nomeasurable binding to HPA-1a-negative αllbβ3 (FIG. 4A). Rapidassociation and slow dissociation indicate that the 26.4 binds stronglyto the HPA-1a antigen. The binding properties of 26.4 recombinantantibodies were identical to the hybridoma-secreted batch (data notshown).

Further, we compared binding properties of the 26.4 to the humanHPA-1a-specific mAb, done B2G1, generated by phage display also from awoman alloimmunized in connection with pregnancy (Griffin H, OuvvehandW., Blood. 1995; 86(12):4430-6). Similar association and dissociationcurves for 26.4 and B2G1 indicate that affinities of the two mAbs are inthe same range (FIG. 5A). Binding affinity of the B2G1 to therecombinant αllbβ3 was measured previously, K_(D)=6×10⁻⁸ (Santoso S,Kroll H, Andrei-Selmer C L, Socher I, Rankin A, Kretzschmar E, et al.Transfusion. 2006;46(5):790-9.).

Next, we assessed binding properties and specificity of the previouslycharacterized mouse mAb, clone SZ21 (Weiss E J, Goldschmidt-Clermont PJ, Grigoryev D, Jin Y, Kickler T S, Bray P F. Tissue Antigens.1995;46(5):374-81.). The SZ21antibody bound both HPA-1a positive andnegative integrin αllbβ3, however, it displayed a higher affinity forHPA-1a as it associated slower and dissociated faster from the HPA-1anegative integrin (FIG. 4B). This binding pattern indicates that SZ21 ispseudospecific for HPA-1a.

MAb 26.4 Displays a Unique Binding Pattern to Integrin αVβ3

As integrin β3 is also part of the vitronectin receptor (αVβ3) weexamined whether or not the HPA-1a-specific mAbs 26.4 and B2G1 couldbind to purified αVβ3. The source and method of purifying the αVβ3integrin is described above. Both mAbs bound to the sensor chip surfacecoupled with αllbβ3 (HPA-1a ) and αVβ3 (FIG. 5A and 5B). However, 26.4bound to αllbβ3 generating 10% more binding response than B2G1.Surprisingly, the difference in binding response was more profound onthe surface coupled with αVβ3: 26.4 generated 42% more binding responsethan B2G1 (FIG. 5C).

Both mAbs dissociated from the αllbβ3 with nearly identical rate; around81% of the bound 26.4 as well as B2G1 remained bound at the end of thedissociation period. However, B2G1 dissociated from the αVβ3 over 50%faster than 26.4; 31.4% of B2G1 compared to 66.8% of 26.4 remained boundat the end of dissociation period (FIG. 5D).

The difference is not attributed to any loss of antigen during theregeneration procedure as the B2G1 samples were run before the 26.4samples over both the αallbβ3 (FIG. 5A) and the αVβ3 (FIG. 5B) surfaces.Furthermore, the results were produced with various antibodyconcentrations, 20, 10 and 5 μg/ml (only 20 μg/ml is shown) and similarresults have been obtained using sensor chip coupled with higher amountsof integrins (data not shown).

Further association/dissociation data is shown in Table 2

TABLE 2 SPR analysis of mAb 26.4 and B2G1 binding to immobilized αIIbβ3and αVβ3. 26.4 B2G1 bound after % bound after % Integrin bounddissociation disso- bound dissociation disso- complex (RU) (RU) ciated(RU) (RU) ciated αIIbβ3 63.1 49.6 21.4 56.1 43.8 22 αVβ3 18.8 12.2 35.111.2 3.3 70.5

Due to the observed difference in binding to αVβ3, it was decided toexamine the relative efficiencies of 26.4 and B2G1at inhibiting thebinding of a third anti-HPA-1a mAb, SZ21, to αllbβ3 and αVβ3 (FIGS. 5Eand 5F). In this set of experiments, mAb 26.4 was more efficient thanB2G1at inhibiting binding of SZ21 to beads coupled with αVβ3 fromtrophoblasts (FIG. 5F). In comparison, there was little difference inthe efficiency of the two mAbs (26.4 and B2G1) at inhibitingSZ21-binding to beads coupled with αllbβ3 from platelets (FIG. 5E).Therefore, although mAbs 26.4 and B2G1appear to bind similarly to HPA-1aon integrin αllbβ3, they differ in binding efficiency to integrin αVβ3.

MAb 26.4 has Inhibitory Effect on Platelet Aggregation

Since integrin heterodimer αllbβ3 is a fibrinogen receptor on platelets,we assessed whether 26.4 affects platelet aggregation (FIG. 6). The 26.4inhibited HPA-1aa platelet aggregation in a concentration-dependantmanner: 15, 35 and 80% inhibition at concentrations of 2, 6 and 12μg/ml, respectively, compared with the aggregation control. Theaggregation control was the individual platelet aggregation triggered byTRAP-6. The individual platelet aggregation was taken as 100%. At thelowest mAb concentration, inhibition of aggregation of the HPA-1a bplatelets was similar to HPA-1a a. The 6 and 12 μg/ml of mAb equallyinhibited aggregation of HPA-1a b platelets by 20%. Importantly, therewas no significant effect of the 26.4 on HPA-1bb platelet aggregation.The 26.4 antibody did not affect platelet function when aggregation wasmeasured in samples without platelet activator (data not shown).Platelet count in samples with added mAb in different concentrations didnot differ from control samples without mAb for each participant (datanot shown). The decrease of platelet aggregation is therefore attributedsolely to the inhibition of platelet function.

MAb 26.4 is Potent in Inducing Platelet Phagocytosis

To assess whether 26.4 can induce platelet phagocytosis, we incubatedfreshly isolated monocytes with 26.4-sensitised CFSE-labelled plateletsand measured the frequence of monocytes with ingested platelets by flowcytometry. MAb 26.4 induced phagocytosis of sensitized HPA-1a-homozygousplatelets in a concentration-dependent manner (FIG. 7A). MAb 26.4G1performed similarly to 26.4IgG3. At concentrations 10, 1and 0.1 μg/mlthe antibodies induced around 90, 70 and 30% phagocytosis, respectively.These % phagocytosis values are the % of monocytes that had internalizedplatelets. The phagocytic activity was close to 10% when 0.01 μg/ml ofthe antibody was used as well as in negative controls. The phagocyticactivity with HPA-1ab platelets was about 20% lower compared to HPA-1aaplatelets (FIG. 7B). The antibodies did not affect phagocytosis ofsensitized HPA-1a -negative platelets. No synergistic effect wasobserved when a 1:1 mixture of 26.4G1and 26.4IgG3 was tested in similarexperiments (data not shown).

Discussion

In the study described herein a recombinant monoclonal antibody specificfor HPA-1a was derived from a single memory B cell. This B cell wasisolated from a woman known to be HPA-1a immunized in connection withpregnancy. This antibody, clone 26.4, has been successfully expressedrecombinantly by transient transfection of human cells. It has beenfound that 26.4 binds strongly to HPA-1a and is highly specific; noreactivity to the HPA-1b allotype was detected, Furthermore, it exhibitsonly a modest inhibitory effect on HPA-1ab platelet aggregation and canopsonize platelets for enhanced monocyte phagocytosis. Thus, mAb 26.4holds potential both for FNAIT prophylaxis and HPA-1a typing.

It has been demonstrated herein by sensitive binding assays that therewas no measurable cross-reactivity of mAb 26.4 with the native HPA-1ballotype. Without wishing to be bound by theory, it is believed thatthis can be attributed to selection of the antibody by the human immunesystem. The difference between the HPA-1 allotypes is a single aminoacid, which is leucine in HPA-1a. MAb 26.4 is obviously not binding toleucine alone. Therefore, one possibility is that the antibody hasaffinity for a surface area that is common to both allotypes and thatthe allogeneic leucine makes the difference between stable binding withit and no binding without. Alternatively, the single amino aciddifference may be associated with a conformational change that in effectcreates a new epitope that the antibody can bind to. In either of theabove cases, the in vivo selection and affinity maturation in the B cellthat gave rise to mAb 26.4 was likely driven towards the highest bindingaffinity to the alloantigen and at the same time maintaining lowcross-reactivity with the HPA-1 b autologous counterpart. In developinganti-HPA-1a antibodies by immunization of mice, a similar pressure toselect for minimal cross-reactivity with HPA-1b will be lacking. This isconsistent with the observations herein of considerable cross-reactivityof the SZ21antibody with HPA-1b while none was detectable with mAb 26.4.Without wishing to be bound by theory, it is believed that anti-HPA-1aantibodies which are able to cross-react with the antigen HPA-1 b (e.g.the antibody SZ21) could cause undesirable immune responses in themother, e.g. accelerate removal of maternal HPA-1bb platelets from theblood circulation causing thrombocytopenia.

Platelet aggregation is central in haemostasis and thrombosis andintegrin αllbβ3 plays a critical role in it. Previous studiesdemonstrated that anti-HPA-1a antibodies had an inhibitory effect onplatelet aggregation and adhesion of αllbβ3 and αVβ3 transfected CHOcells to fibrinogen (Joutsi-Korhonen L, Preston S, Smethurst P A,Ijsseldijk M, Schaffner-Reckinger E, Armour K L, et al. Thrombosis andHaemostasis, 2004;91(4):743-54, and Kroll H, Penke G, Santoso S.Thrombosis and Haemostasis. 2005;94(12):1224-9.).

The mechanism of fetal platelet destruction by maternal anti-HPA-1aantibodies is not completely understood. Without wishing to be bound bytheory it is speculated that IgG sensitized fetal platelets are removedfrom circulation via FcyR-mediated phagocytosis by mononuclearphagocytes in the spleen and liver and possibly by granulocytes. Oneapplication of anti-HPA-1a mAbs is as a prophylaxis against HPA-1aallommunization. One of the proposed mechanisms of prevention ofimmunization against the RhD-antigen is by removing fetal red bloodcells from maternal circulation via phagocytosis of anti-RhDIgG-opsonized red blood cells. Similarly, and again without wishing tobe bound by theory, it is hypothesised that HPA-1a immunization may beprevented by anti-HPA-1a antibodies by sensitizing fetal platelets whichwill then be removed from maternal circulation by phagocytes. We havedemonstrated in a human in vitro system that mAb 26.4 (Iga1 and IgG3)can induce phagocytosis of HPA-1a -positive platelets.

As described above, a notable difference between the 26.4 antibody andthe B2G1 antibody is that 26.4 binds more stably to trophoblast-derivedαvβ3 and is more efficient at inhibiting binding of anti-HPA-1aantibodies (SZ21) to αVβ3. In terms of prophylactic and therapeuticpotential, stable binding to HPA-1a on trophoblasts may be anadvantageous property. It is believed that HPA-1a on αVβ3 expressed ontrophoblast cells could initiate an alloimmune response in the mother(Vanderpuye O A, et al., (1991), Biochem J 280 (Pt 1): 9-17; Kumpel etal. (2008), Transfusion 48: 2077-86). Without wishing to be bound bytheory, the stable binding of 26.4 to αVβ3 derived from placenta couldaccelerate removal of cells and material expressing this antigen fromthe maternal circulation and thereby prevent alloimmunization. Again,without wishing to be bound by theory, an additional mechanism could bemasking of epitopes and in effect preventing HPA-1a-specific B cellsfrom binding antigen and thereby prevent their activation. Removal fromthe circulation could also prevent activation of such B

In conclusion, we have developed a novel HPA-1a-specific antibodyderived from a single B cell of a woman HPA-1a alloimmunized inconnection with pregnancy. The antibody has no detectable crossreactivity with the HPA-1b allotype. The recombinant version of thisantibody may be used as a diagnostic reagent to identify the individualsat risk of HPA-1a immunization as well as a prophylactic reagent toprevent FNAIT and/or as a therapeutic agent to treat FNAIT.

Example 2

A Novel Human Recombinant Monoclonal HPA-1a-Specific Antibody is aUseful Tool for Diagnostics in Fetal and Neonatal AlloimmuneThrombocytopenia

Introduction

Currently, there is no safe and effective prevention or treatment of thecondition and the majority of FNAIT cases are diagnosed after birth of aseverely thrombocytopenic child. It will be important to identify womenat risk of immunization which could benefit from the prophylactictreatment.

Several prospective studies found that high levels of maternalanti-HPA-1a antibodies correlate with low platelet count in the newborn.Therefore, quantitation of anti-HPA-1a antibodies can be used as apredictive factor of the degree of thrombocytopenia in the newborn.Currently used reference material for anti-HPA-1a antibody quantitationwas prepared by the National institute of Biological Standards andControl (NIBSC). This NIBSC standard contains plasma from six HPA-1aimmunized donors and its supply is dependent on the availability of suchdonors.

In the present studies, a novel HPA-1a-specific human recombinantmonoclonal antibody, clone 26.4, has been generated. This mAb can beused as a reagent for HPA-1 phenotyping as well as a standard forquantitation of anti-HPA-1a antibodies.

The aim of the study was to evaluate whether the human HPA-1a -specificmAb, clone 26.4, can distinguish HPA-1a and HPA-1b antigens in a wholeblood flow cytometry assay. The second aim was to evaluate whether thismAb can be used as a standard for quantitative MAIPA assay.

Materials and Methods

Donor Blood Samples

Peripheral blood was obtained from random healthy blood donor volunteersthat have agreed to donate samples that could be used for researchpurposes (Blood Bank, University Hospital of North Norway). The HPA-1aimmunized women donated blood after signing a written informed consent(study was approved by Regional Committee for Medical Research Ethics,North-Norway, approval no: 2009/1585).

Antibodies

An HPA-1a -specific mAb IgG1, clone 26.4, was generated byimmortalization of antigen-specific memory B cells from a woman HPA-1aimmunized in connection with pregnancy and expressed recombinantly. TheIgG1 was purified from cell culture supernatant by 40% saturatedammonium sulphate precipitation followed by Protein G affinitychromatography (Protein G Sepharose 4 FastFlow, GE Healthcare).

The established WHO international reference reagent for quantitation cfanti-HPA-1a antibodies was obtained from the National Institute forBiological Standards and Controls (NIBSC, code 03/152) (Allen D, et al.Vox Sanguinis. 2005;89(2):100-4).

HPA-1 Genotyping

Donor samples were HPA-1 genotyped using TagMan 5′ nuclease assay asdescribed previously (Bogert P, McBride S, Smith G, Dugrillon A, KlüterH, Ouwehand W H, et al. Transfusion. 2005;45(5):654-9).

HPA-1 Phenotyping by Whole Blood Flow Cytometry

Purified mAb 26.4 IgG1 was conjugated with Alexa Fluor 488 fluorescentdye according to the manufacturer instructions (Molecular Probes). Thedegree of labeling (DOL) was calculated using formula: mole dye/moleprotein. Forty microliters of the mAb diluted in PBS containing 0.3%EDTA and 0.2% BSA were added to 10 pl EDTA-anticoagulated whole bloodand incubated for 10 minutes at RT in the dark. After adding 0.5 ml ofPBS 0.3% EDTA 0.2% BSA buffer the samples were analyzed by flowcytometry (FACSCanto, BD Biosciences). HPA-1aa and HPA-1bb plateletswere used as controls. Median FITC fluorescence intensities (MFI) of thecontrols and each sample were recorded, Flow cytometry data was analysedby FlowJo software (TreeStar, Ashland, Oreg., USA). The blood sampleswere HPA-1 phenotyped within 10 days of storage, as older samples wereviscous and difficult to pipette.

Probing mAb 26.4 as a Standard for Anti-HPA-1a Antibody Quantitation byMAIPA

Purified mAb 26.4 IgG1 was buffer exchanged into phosphate-bufferedsaline (PBS) containing 0.02% sodium azide and bovine serum albumin(BSA) was added to a concentration of 0.5%. The concentration of mAb wasdetermined by ELISA as described in Example 1. The mAb26.4 wasquantified by monoclonal antibody immobilization of platelet antigens(MAIPA) assay with mouse anti-human CD61, clone Y2/51 (Dako, Denmark),used as a capture antibody (Killie M K, Salma W, Bertelsen E, Skogen B,Husebekk A. 2010;43(2)1 49-54.). MAIPA was originally described byKiefel et at. (supra); the modified rapid protocol with the reagents isrecommended by NIBSC (Modified Rapid MAIPA Assay, http://www.nibsc.org,and Kjeldsen-Kragh J, Killie M K, Tomter G. Golebiowska E. Randen I,Hauge R, et al. Blood. 2007;110(3):833-9).

Replicate doubling dilutions (1:8 1:512) of the international polyclonalanti-HPA-1a NIBSC standard together with the mAb 26.4 preparation wereused to create a linear standard curve. Four plasma samples withdifferent levels of anti-HPA-1a antibodies were tested against HPA-1aaplatelets. The levels of specific antibodies in the samples werecalculated using linear regression equation.

To assess the intra-assay variability (accuracy) the samples were testedin triplicates. Intra assay coefficient of variation (intra assay CV) isthe average of the individual CVs and calculated using formula: %CV=Meanof SD×100/Mean;

To assess the inter-assay variability (reproducibility) the assay wasrepeated three times. It is expressed by inter assay coefficient ofvariation (inter assay CV) and calculated following formula: %CV=SD ofMean×100/Mean.

Results

MAb 26.4 IgG1 is a Potential HPA-1a Phenotyping Reagent

To test whether mAb 26.4 IgG1 can distinguish between HPA-1a and HPA-1bplatelets in whole blood samples, first, we fluorescently labeled themAb with AlexaFuor 488 dye. The degree of labeling (DOL) was calculatedto be around 3 (recommended by the manufacturer optimal DOL should be ˜2fluorophores per antibody). We determined the amount of the AlexaFuor488-conjugated mAb that allowed us to distinguish HPA-1a -positive from-negative samples (FIG. 8),

We phenotyped 143 donor blood samples (random donor samples togetherwith samples from the individuals with known HPA-1 genotype, Table A).

TABLE A HPA-1 genotyped and phenotyped donor blood samples. Total numberof samples HPA-1aa HPA-1ab HPA-1bb 143 98 30 15

The recorded median FITC fluorescence intensities (MFI) of all theHPA-1a-positive samples were significantly higher (5 times or more) thanthe MFI of the HPA-1a-negative samples. All the blood samples were HPA-1genotyped. In the collection of tested blood samples, we detected nophenotype-genotype discrepancies.

MAb 26.4 IgG1 can be used as a Standard for Detection and Quantitationof Anti-HPA-1a Antibodies by MAIPA Assay

To evaluate the use of mAb 26.4 IgG1as a standard in quantitative MAIPAwe aligned MAb 26.4 IgG1 with the international polyclonal anti-HPA-1aNIBSC standard. At a concentration 5 μg/ml the mAb had anti-HPA-1aactivity corresponding to 100 IU/ml.

We compared plots generated by mean absorbance values for replicatedoubling dilutions of the international polyclonal anti-HPA-1a NIBSCstandard and the mAb 26.4 IgG1 standard in MAIPA assay. The linearityand range of the two standards were comparable (FIG. 9). The linearportions of the plots were used to determine the anti-HPA-1a antibodylevels of the samples. The mean values of anti-HPA-1a activities insamples A, B, C and D measured in three assays are presented in FIG. 10.

Intra assay variation describes the variation of results within a dataset obtained from one experiment (accuracy). The intra-assay CVs (n=12)were calculated to be around 6% for both, NIBSC and mAb 26.4. The interassay variation describes the variation of results obtained fromrepeated experiments (reproducibility). The inter-assay CVs (n=3) werecalculated to be around 9% and 10% for NIBSC and mAb 26.4 as standardsrespectively.

Discussion

There is a demand for a reagent that could be used to establish a simpleand reliable technique to identify HPA-1a-negative individuals. TheHPA-1a genotyping techniques are reliable but time consuming or requiresophisticated equipment. The commercially available ELISA-based assay isexpensive and unreliable due to false positive results. The twopreviously published flow cytometry-based assays rely on SZ21antibody.The SZ21 mAb is pseudospecific to the HPA-1a; it binds toHPA-1a-negative platelets in increasing antibody concentrations. Ahighly specific for HPA-1a mAb would be advantageous for the phenotypingassays reducing the probability of false positive results.

To validate whether a novel human HPA-1a-specific mAb 26.4 candistinguish HPA-1a from b allotype in whole blood samples, we HPA-1phenotyped 143 whole blood samples using the fluorophore-conjugated 26.4to and found no phenotype-genotype discrepancies. The whole blood flowcytometry-based HPA-1 phenotyping using this mAb is a rapid and reliabletechnique suitable for screening purposes.

Phenotyping may be supplemented with genotyping of the identifiedHPA-1a-negative samples.

Quantitation of the anti-HPA-1a antibodies has a predictive value indiagnosis of FNAIT. The available from NIBSC anti-HPA-1a referencematerial (NIBSC code: 03/152) consists of pooled plasma from severalHPA-1a immunized donors and its supply is dependent on the availabilityof such donors. We reasoned that the recombinant monoclonal antibodywould facilitate an unlimited supply of a standardized and relativelyinexpensive reagent. We found that the 26.4 shows high accuracy andreproducibility, similar to the NIBSC reference material, when used as astandard for quantitation of samples with different anti-HPA-1a antibodylevels.

Example 3

MAb 26.4 Inhibits Binding of Polyclonal Anti-HPA-1a IgG to Platelets

MAIPA Inhibition Assay

MAb 26.4 F(ab′)₂ fragment was prepared using Pierce F(ab′)₂ PreparationKit (Pierce, Appleton, Wis.). The purified F(ab′)₂ fragmentconcentration (0.7 mg/ml) was determined by spectrophotometry from theabsorbance at 280 nm using an extinction coefficient of 1.4 L×g⁻¹×cm⁻¹.The ability of 26.4 to block binding of polyclonal maternal anti-HPA-1aIgG antibodies was evaluated by a modified adaptation of the MAIPAtechnique (Griffin H, Ouwehand W. 1995. Blood 86: 4430-6). Briefly,HPA-1a homozygous fresh platelets (2×10⁷) were incubated with 50 μl of26.4 F(ab′)₂ for lh at RT before adding 100 μl of diluted 1:10 serumsamples for 15 min. Further, the MAIPA assay was performed as describedpreviously (Kiefel V et al. 1987. Blood 70: 1722-6; Killie M K et al.2010. Transfusion and Apheresis Science 43: 149-54). We tested a panelof 10 donor serum samples with anti-HPA-1a activity ranging from 10 to150 IU/ml as measured by quantitative MAIPA (Killie M K et al. 2010.Transfusion and Apheresis Science 43: 149-54).

One potential therapeutic use of mAb 26.4 would involve blocking accessof pathogenic anti-HPA-1a antibodies to fetal platelets. Therefore, wetested the capacity of 26.4 to inhibit binding of maternal polyclonalanti-HPA-1a IgG using the MAIPA technique. Binding to HPA-1a homozygousplatelets in 10 out of 10 samples was considerably inhibited afterpreincubation of platelets with 26.4 F(ab′)₂ fragment. The inhibitionranged from 65% to 100% at a highest fragment concentration of 35 μg in50 μl volume (FIG. 11). GraphPad Prism 5 software (San Diego, Calif.)was used to present MAIPA inhibition assay data.

Without wishing to be bound by theory, it is believed that antibodieswhich have a reduced or abolished effector function (e.g, a F(ab′)₂fragment of 26.4) would be useful in FNAIT treatment as such antibodieswould cross the placenta and bind fetal platelets, thereby hinderingbinding of functional maternal anti-HPA-1a IgG antibodies and protectingfetal tissues and platelets from potentially damaging maternalanti-HPA-1a antibodies. The demonstration that mAb 26.4 can efficientlyblock maternal polyclonal HPA-1a-specific IgG from various donors frombinding platelets suggests that the mAb could also interfere withbinding to receptors on HPA-1a -specific B cell clones in womensusceptible to immunization.

Example 4

Domain Deletion Peptide ELISA

Anti-HPA-1a antibodies are heterogeneous in their footprint on the β₃integrin and are categorized as type I and type II antibodies (Liu L Xet al., Blood, 1996;88(9):3601-7; Valentin N et al., Blood,1995;85(11);3028-33; Stafford P et al. Journal of Thrombosis andHaemostasis, 2008;6(2):366-75). Type I antibodies bind to the residueswithin the plexin/semaphorin/integrin (PSI) domain, the first 54residues of the β₃ integrin which contain the HPA-1 polymorphism atposition 33. The epitope of the type II antibodies spans to the residuesdistant from the PSI domain—the hybrid and epidermal growth factor (EGF)domains.

It was decided to test whether 26.4 epitope is constrained to PSI domainor spans several domains of the β₃ integrin. To study this, thedomain-deletion peptide ELISA technique described previously wasemployed (Stafford P et al. Journal of Thrombosis and Haemostasis,2008;6(4;366-75).

Materials

Antibodies

Integrin β₃-specific murine mAb clones Y2/51 (Beckman Coulter, Pasadena,Calif.) and SZ21 (Dako, Glostrup, Denmark) were used. Integrinαllb-specific mAb clone SZ22 (Beckman Coulter, Pasadena, Calif.) wasused. Human mAb specific for HPA-1a, clone B2G1 was isolated frommaternal B cells of a case of FNAIT using phage display (Griffin H,Ouwehand W, Blood 1995;86(12):4430-6) and produced recombinantly (Garneret al., British Journal of Haematology, 2000;108(2);440-7) (kindlyprovided by Cedric Ghevaert, Department of Hematology, School ofClinical Medicine, University of Cambridge, UK). MAb 26.4 derived from asingle B cell isolated from a woman HPA-1a -immunized in connection withpregnancy (described herein). Horseradish peroxidase (HRP)-conjugatedgoat anti-mouse IgG and HRP-conjugated goat anti-human IgG (JacksonImmunoResearch Laboratories, West Grove, Pa.) were used as secondaryantibodies.

Recombinant Domain-Deleted Peptides

The following peptides were used: ΔβA-Leu33, ΔβpA-Pro33, PSI-Leu33, andGPVI (hDID2) as a negative control (peptides kindly provided by RoseyMushens, International Blood Group Reference Laboratory, NHS Blood andTransplant, Filton, Bristol, UK; Winnie Chong, Department ofHistocompatibility and Immunogenetics, NHS Blood and Transplant,Colindale Avenue, London, UK; Willem H Ouwehand, University of Cambridge& Wellcome Trust Sanger Institute, NHS Blood and Transplant, UK;Stafford P et al. describe these peptides in Journal of Thrombosis andHaemoslasis, 2008;6(2):366-75). CaM-binding peptide N9A coupled to BSAwas kindly provided by Peter Smethurst and Nicola Foad (described bySmethurst PA et al., Blood 2004;103(3):903-11).

Methods

Cloning, expression and purification of the recombinant domain-deletionpeptides with calmodulin (CaM) tag is described in Stafford et al.(Journal of Thrombosis and Haemostasis, 2008;6(2):366-75). ELISA wasperformed as described previously (Abou-Chaker K et al., Tissue Antigens2009;73(3);242-4), Briefly, the β₃ peptides were immobilized to ELISAplates via CaM-binding peptide N9A coupled to BSA (Smethurst P A et al.,Blood 2004;103(3):903-11). Murine and human mAbs were used atconcentrations of 1 and 10 μg/ml. MAb binding was detected byHRP-conjugated goat-anti-mouse IgG or HRP-conjugated goat-anti-humanIgG. Absorbance at 492 nm was read on an microplate photometer(Multiskan E X, Thermo Scientific, Waltham, Mass.). Each sample wastested in duplicate and average absorbance values were used to generatethe graph (FIG. 12 and FIG. 13).

Results

Binding of the murine mAbs, clones Y2/51and SZ21, to domain-deletionpeptides was published previously (Stafford P et al., Journal ofThrombosis and Haemostasis, 2008;6(2):366-75) and was used as a systemcontrol. MAb Y2/51at concentrations of 1and 10 μg/ml bound themulti-domain peptide ΔβA, Leu33 and Pro33, variants. MAb SZ21at 1 μg/mlbound to ΔβA-Leu33, when binding to ΔβA-Pro33 and PSI-Leu33 generatedrelatively low response. MAb SZ21at 10 μg/ml bound multi-domain peptidesΔβA, independently on Leu33 or Pro33 variant, as well as a single-domainpeptide PSI-Leu33. None of the mAbs bound to the control peptide. Theresults were consistent with the previously published (Stafford P et al.Journal of Thrombosis and Haemostasis, 2008;6(2):366-75). MAb SZ22(specific to αllb, CD41) was used as a murine mAb negative control anddid not bind neither of the peptides (data not shown).

MAb 26.4 bound exclusively to the multi-domain peptide ΔβA-Leu33; nobinding to the ΔβA-Pro33, single-domain peptide PSl-Leu33 or peptidenegative control was observed. MAb B2G1 had an identical bindingpattern, consistent with the previously published results (Stafford P etal., Journal of Thrombosis and Haemostasis, 2008;6(2):366-75).

The results described above suggest that epitope of the 26.4 spansseveral domains of β₃ integrin, and 26.4 is a type Il anti-HPA-1aantibody.

1. A method of preventing immunization with HPA-1a in an HPA-1a-negativesubject, the method comprising administering to the subject an effectiveamount of an anti-HPA-1a antibody, wherein the antibody comprises avariable region light chain (VL) comprising: (a) a VL complementaritydetermining region (CDR)1 that has the amino acid sequence of SEQ ID NO:8; (b) a VL CDR2 that has the amino acid sequence of SEQ ID NO: 9; and(c) a VL CDR3 that has the amino acid sequence of SEQ ID NO: 10; andwherein the antibody comprises a variable region heavy chain (VH)comprising: (d) a VH CDR1 that has the amino acid sequence of SEQ ID NO:5; (e) a VH CDR2 that has the amino acid sequence of SEQ ID NO: 6; and(f) a VH CDR3 that has the amino acid sequence of SEQ ID NO:
 7. 2. Amethod of preventing fetal and neonatal alloimmune thrombocytopenia(FNAIT) in a fetus of a subject, the method comprising administering tothe subject an effective amount of an anti-HPA-1a antibody, wherein theantibody comprises a variable region light chain (VL) comprising: (a) aVL complementarity determining region (CDR)1 that has the amino acidsequence of SEQ ID NO: 8; (b) a VL CDR2 that has the amino acid sequenceof SEQ ID NO: 9; and (c) a VL CDR3 that has the amino acid sequence ofSEQ ID NO: 10; and wherein the antibody comprises a variable regionheavy chain (VH) comprising: (d) a VH CDR1 that has the amino acidsequence of SEQ ID NO: 5; (e) a VH CDR2 that has the amino acid sequenceof SEQ ID NO: 6; and (f) a VH CDR3 that has the amino acid sequence ofSEQ ID NO: 7; wherein the subject is an HPA-1a-negative woman.
 3. Amethod of treating fetal and neonatal alloimmune thrombocytopenia(FNAIT) in a fetus of a subject, the method comprising administering tothe subject an effective amount of an anti-HPA-1a antibody, wherein theantibody comprises a variable region light chain (VL) comprising: (a) aVL complementarity determining region (CDR)1 that has the amino acidsequence of SEQ ID NO: 8; (b) a VL CDR2 that has the amino acid sequenceof SEQ ID NO: 9; and (c) a VL CDR3 that has the amino acid sequence ofSEQ ID NO: 10; and wherein the antibody comprises a variable regionheavy chain (VH) comprising: (d) a VH CDR1 that has the amino acidsequence of SEQ ID NO: 5; (e) a VH CDR2 that has the amino acid sequenceof SEQ ID NO: 6; and (f) a VH CDR3 that has the amino acid sequence ofSEQ ID NO: 7; wherein the subject is an HPA-1a-negative woman.
 4. Amethod of treating fetal and neonatal alloimmune thrombocytopenia(FNAIT) in a neonatal subject, the method comprising administering tothe subject an effective amount of an anti-HPA-1a antibody, wherein theantibody comprises a variable region light chain (VL) comprising: (a) aVL complementarity determining region (CDR)1 that has the amino acidsequence of SEQ ID NO: 8; (b) a VL CDR2 that has the amino acid sequenceof SEQ ID NO: 9; and (c) a VL CDR3 that has the amino acid sequence ofSEQ ID NO: 10; and wherein the antibody comprises a variable regionheavy chain (VH) comprising: (d) a VH CDR1 that has the amino acidsequence of SEQ ID NO: 5; (e) a VH CDR2 that has the amino acid sequenceof SEQ ID NO: 6; and (f) a VH CDR3 that has the amino acid sequence ofSEQ ID NO:
 7. 5. A method of inhibiting aggregation of HPA-1a-positiveplatelets, the method comprising contacting the HPA-1a-positiveplatelets with an anti-HPA-1a antibody, wherein the antibody comprises avariable region light chain (VL) comprising: (a) a VL complementaritydetermining region (CDR)1 that has the amino acid sequence of SEQ ID NO:8; (b) a VL CDR2 that has the amino acid sequence of SEQ ID NO: 9; and(c) a VL CDR3 that has the amino acid sequence of SEQ ID NO: 10; andwherein the antibody comprises a variable region heavy chain (VH)comprising: (d) a VH CDR1 that has the amino acid sequence of SEQ ID NO:5; (e) a VH CDR2 that has the amino acid sequence of SEQ ID NO: 6; and(f) a VH CDR3 that has the amino acid sequence of SEQ ID NO:
 7. 6. Amethod of inhibiting binding of a first anti-HPA-1a antibody to integrinαVβ3, the method comprising contacting integrin αVβ3 with a secondanti-HPA-1a antibody, wherein the second anti-HPA-1a antibody comprisesa variable region light chain (VL) comprising: (a) a VL complementaritydetermining region (CDR)1 that has the amino acid sequence of SEQ ID NO:8; (b) a VL CDR2 that has the amino acid sequence of SEQ ID NO: 9; and(c) a VL CDR3 that has the amino acid sequence of SEQ ID NO: 10; andwherein the antibody comprises a variable region heavy chain (VH)comprising: (d) a VH CDR1 that has the amino acid sequence of SEQ ID NO:5; (e) a VH CDR2 that has the amino acid sequence of SEQ ID NO: 6; and(f) a VH CDR3 that has the amino acid sequence of SEQ ID NO:
 7. 7. Themethod according to claim 1, wherein the VL has the amino acid sequenceof SEQ ID NO: 4, or a sequence having at least 80% sequence identitythereto.
 8. The method according to claim 1, wherein the VH has theamino acid sequence of SEQ ID NO: 3, or a sequence having at least 80%sequence identity thereto.
 9. The method according to claim 1, whereinthe VL has the amino acid sequence of SEQ ID NO: 4, and wherein the VHhas the amino acid sequence of SEQ ID NO:
 3. 10. The method according toclaim 1, wherein the antibody is a full-length IgG antibody.
 11. Themethod according to claim 10, wherein the antibody has a heavy chainthat comprises the amino acid sequence of SEQ ID NO: 21 or a sequencehaving at least 80% sequence identity thereto, and/or a light chain thatcomprises the amino acid sequence of SEQ ID NO: 22 or a sequence havingat least 80% sequence identity thereto.
 12. The method according toclaim 10, wherein the antibody has a heavy chain that comprises theamino acid sequence of SEQ ID NO: 25 or a sequence having at least 80%sequence identity thereto, and/or a light chain that comprises the aminoacid sequence of SEQ ID NO: 26 or a sequence having at least 80%sequence identity thereto.
 13. The method according to claim 10, whereinthe antibody: (i) has a heavy chain that comprises the amino acidsequence of SEQ ID NO:21and a light chain that comprises the amino acidsequence of SEQ ID NO:22, or (ii) has a heavy chain that comprises theamino acid sequence of SEQ ID NO:25 and a light chain that comprises theamino acid sequence of SEQ ID NO:26.
 14. The method according to claim1, wherein the antibody has an abolished effector function.
 15. Themethod according to claim 1, wherein the antibody is not fucosylated.16. The method according to claim 5, wherein the method comprisesadministering the antibody to a subject in need thereof.
 17. The methodaccording to claim 6, wherein the method comprises administering theantibody to a subject in need thereof.